Heparosan-producing bacterium and heparosan manufacturing method

ABSTRACT

A method for producing heparosan is provided. Heparosan is produced by culturing an  Escherichia  bacterium having a heparosan-producing ability and modified so that expression of one or more genes, such as rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, rfaH, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, lepB, rnc, era, dapA, gcvR, bcp, hyfA, rpoE, nadB, yfiC, srmB, g1414, g1413, nuoE, nuoF, nuoG, glmZ, hemY, hemX, hemD, rlmL, artQ, artM, artJ, rlmC, ybjO, yejO, yejM, yejL, rpoS, ygbN, ygbM, ygbL, g3798, g3797, g3796, g3795, g3794, g3793, g3792, ryjA, soxR, soxS, yjcC, yjcB, efeU, and efeO, is/are increased in a medium, and collecting heparosan from the medium.

This application is a Continuation of, and claims priority under 35U.S.C. § 120 to, International Application No. PCT/JP2014/076357, filedOct. 2, 2014, and claims priority therethrough under 35 U.S.C. § 119 toJapanese Patent Application No. 2013-207003, filed Oct. 2, 2013,Japanese Patent Application No. 2013-259620, filed Dec. 16, 2013,Japanese Patent Application No. 2013-259621, filed Dec. 16, 2013, andJapanese Patent Application No. 2014-039250, filed Feb. 28, 2014, theentireties of which are incorporated by reference herein. Also, theSequence Listing filed electronically herewith is hereby incorporated byreference (File name: 2016-03-28T_US-545_Seq_List; File size: 570 KB;Date recorded: Mar. 28, 2016).

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a heparosan-producing bacterium and amethod for producing heparosan.

Brief Description of the Related Art

Heparosan (also referred to as N-acetylheparosan) is a polysaccharideconstituted by a repetition structure of a disaccharide having aglucuronic acid (GlcUA) residue and an N-acetyl-D-glucosamine (GlcNAc)residue [→4)-β-GlcUA-(1→4)-α-GlcNAc-(1→].

In nature, heparosan is produced by the Escherichia coli K5 strain andthe Pasteurella multocida type D strain as a capsular polysaccharide(Lindahl U. et al. (1998) J. Biol. Chem., 273(39):24979-24982). Theseheparosan-producing bacteria are pathogenic and cause urinary tractinfections, atrophic rhinitis, etc. in mammals.

In the Escherichia coli K5 strain, two kinds of glucosyltransferases,which are heparosan synthetases, and six kinds of heparosan effluxcarriers are required for the biosynthesis of heparosan. That is, GlcNAcand GlcUA are first alternately added to a non-reducing end of the sugarchain by the glucosyltransferases (KfiA and KfiC), and the heparosanchain is thereby extended (Hodson N. et al. (2000) J. Biol. Chem.,275(35):27311-27315). Then, the heparosan chain is transported to thecell surface by the heparosan efflux carriers, which include KpsC, KpsD,KpsE, KpsM, KpsS, and KpsT (McNulty C. et al. (2006) Mol. Microbiol.,59(3):907-22). It is thought that the heparosan chain is fixed to aphosphatidic acid molecule in the outer membrane of Escherichia coli onthe cell surface through lipid substitution at the reducing end (JannB., Jann K. (1990) Curr. Top Microbiol. Immunol., 150:19-42).

In the Escherichia coli K5 strain, the heparosan synthetase genes andthe heparosan efflux carrier genes form a cluster on the chromosome. Thecluster is divided into regions 1 to 3, and region 2, located at thecenter of the cluster, encodes the four proteins including the heparosansynthetases, KfiA, KfiB, KfiC, and KfiD.

The Pasteurella multocida type D strain has PmHS1, which acts as aheparosan synthetase (glucosyltransferase) (Kane T. A. et al. (2006) J.Biol. Chem., November 3; 281(44):33192-33197). PmHS1 has active domainshomologous to both KfiA and KfiC of the Escherichia coli K5 strain, andit catalyzes a polymerization reaction using both UDP-glucuronic acidand UDP-N-acetylglucosamine as substrates. However, to date, noheparosan efflux carriers of the Pasteurella multocida type D strainhave been eluciated.

Heparin is one of anticoagulants, and is useful in therapeutictreatments of thromboembolism and disseminated intravascular coagulation(DIC), prevention of blood coagulation during artificial dialysis andextracorporeal circulation, and so forth. Heparosan is a sugar chainstructure of heparin, and can be converted into a heparin-likepolysaccharide through such steps as deacetylation, epimerization,sulfation, and molecular weight adjustment (Lindahl U. et al. (2005) J.Med. Chem., 48(2):349-352 and Zhang Z. et al. (2008) Journal of theAmerican Chemical Society, 130(39):12998-13007).

Heparin exhibits an anticoagulant activity through activation ofantithrombin III, which is an anticoagulant. Antithrombin III binds tothe active serine moieties of thrombin, Xa factor (active type of Xfactor), and other serine proteases to inhibit them. Thrombin is a bloodcoagulation factor, and the Xa factor is a factor involved in thematuration of thrombin. Heparin binds to this antithrombin III to changethe structure thereof, and thereby activates the inhibitory activity.Thrombin shows higher affinity for the heparin-antithrombin-III complexcompared with the Xa factor.

Low molecular weight heparins having an average molecular weight of 4000to 6000 Da, which are obtainable by enzymatic or chemical treatments ofheparin and fractionation, show less adverse reaction of hemorrhage, andfrequency of use thereof is increasing in recent years. Since the lowmolecular weight heparins have a short sugar chain length, they canbarely bind with thrombin, although they can bind with antithrombin III.For the inhibition of thrombin by the heparin-antithrombin III complex,binding of thrombin to heparin is necessary, but for the inhibition ofthe Xa factor by the heparin-antithrombin III complex, binding of the Xafactor to heparin is unnecessary. Therefore, the low molecular weightheparins hardly inhibit the activity of thrombin, but can inhibit theactivity of the Xa factor.

Most of the currently available heparin preparations utilize extracts ofporcine intestinal mucosa. However, in 2008, a fatal accident occurredas a result of contamination of impurities, and therefore the productionand development of quality-controlled non-animal heparin wasinvestigated.

It has recently been demonstrated through laboratory scale research thatheparosan obtained from the Escherichia coli K5 strain can beenzymatically converted into a heparin-like anticoagulant polysaccharide(Lindahl U. et al. (2005) J. Med. Chem., 48(2):349-352 and Zhang Z. etal. (2008) Journal of the American Chemical Society,130(39):12998-13007). Furthermore, heparosan can also be utilized foruses other than heparin manufacture (WO2009/014559).

Large scale production of heparosan using Escherichia coli K5 is beinginvestigated, and it has been reported that 15 g/L of heparosan wasproduced in a 7-L fermentation tank (Wang Z. et al. (2010) Biotechnol.Bioeng., 107(6):964-973, Japanese Patent Laid-open (Kohyo) No.2013-503606). In order to supply heparosan on an industrial scale as araw material of heparin production, it must be scaled up to the order of100,000 L, but there are issues that must be resolved, particularlyconcerning improving the substrate consumption rate, increasing theoxygen supply in fermentation tank, etc.

Furthermore, a heparosan-producing bacterium produced from anonpathogenic Escherichia coli BL21(DE3) host has very recently beenreported (Zang C. et al. (2012) Metabolic Engineering, 14(5):521-527).That is, in flask culture of the BL21 strain that had been introducedwith an expression vector pETDuet-1 carrying the four heparosanbiosynthesis genes, kfiA, kfiB, kfiC, and kfiD, which constitute region2 of the Escherichia coli K5 strain, production of 334 mg/L of heparosanwas confirmed.

Although the factors required for the heparosan production have beenelucidated, factors that improve heparosan-producing ability of aheparosan-producing bacterium have not been previously reported.

SUMMARY OF THE INVENTION

Aspects to be Achieved by the Invention

Aspects of the present invention are to develop a novel technique forimproving heparosan-producing ability of bacteria, and thereby providean efficient method for producing heparosan.

It has been found that, by increasing the expression of one or more ofthe genes depicted in Tables 1 to 3 in bacteria having aheparosan-producing ability, the heparosan-producing ability isimproved.

TABLE 1 Gene Function of gene product rbsR DNA-binding transcriptionalrepressor of ribose metabolism rbsK enzyme; Degradation of smallmolecules: Carbon compounds rbsB D-ribose transporter subunit hsrA innermembrane protein, multidrug efflux pump glgB 1,4-alpha-glucan branchingenzyme glgX glycogen debranching enzyme micF Regulatory antisense sRNAaffecting ompF expression; member of soxRS regulon rcsD phosphotransferintermediate protein in two-component regulatory system with RcsBC rcsBDNA-binding response regulator in two-component regulatory system withRcsC and YojN ybiX conserved protein ybiI DksA-type zinc finger protein,function unknown ybiJ predicted protein ybiC predicted dehydrogenaseybiB predicted transferase/phosphorylase rfaH transcriptionalantiterminator nusG transcription terminator

TABLE 2 Gene Function of gene product pcoE Probable copper-bindingprotein pcoE precursor pcoS Probable sensor protein pcoS pcoRTranscriptional regulatory protein pcoR yhcN conserved protein yhcOpredicted barnase inhibitor aaeB p-hydroxybenzoic acid efflux systemcomponent aaeA p-hydroxybenzoic acid efflux system component aaeXmembrane protein of efflux system g1455 hypothetical protein alpAPredicted transcriptional regulator g1453 Haemolysin expressionmodulating protein yrbA predicted DNA-binding transcriptional regulator,BolA family mlaB ABC transporter maintaining OM lipid asymmetry,cytoplasmic STAS component mlaC ABC transporter maintaining OM lipidasymmetry, periplasmic binding protein mlaD ABC transporter maintainingOM lipid asymmetry, anchored periplasmic binding protein mlaE ABCtransporter maintaining OM lipid asymmetry, inner membrane permeaseprotein mlaF ABC transporter maintaining OM lipid asymmetry, ATP-bindingprotein yrbG predicted calcium/sodium: proton antiporter norW NADH:flavorubredoxin oxidoreductase ybjI FMN and erythrose-4-P phosphataseybjJ predicted transporter ybjK predicted DNA-binding transcriptionalregulator rybB sRNA effector of ompC and ompW mRNA instability; requiresHfq thrB homoserine kinase thrA fused aspartokinase I and homoserinedehydrogenase I thrL thr operon leader peptide yjtD predicted rRNAmethyltransferase yjjY predicted protein fruA fused fructose-specificPTS enzymes: IIBcomponent/IIC components psuK pseudouridine kinase ytfTpredicted sugar transporter subunit: membrane component of ABCsuperfamily yjfF predicted sugar transporter subunit: membrane componentof ABC superfamily fbp fructose-1,6-bisphosphatase I yagU inner membraneprotein, DUF1440 family paoA PaoABC aldehyde oxidoreductase, 2Fe—2Ssubunit paoB PaoABC aldehyde oxidoreductase, FAD-containing subunit gsiCglutathione transporter, permease component, ABC superfamily gsiDglutathione transporter, permease component, ABC superfamily yliEpredicted cyclic-di-GMP phosphodiesterase, inner membrane protein irp2non-ribosomal peptide synthase irp1 non-ribosomal peptide synthase bhsAbiofilm, cell surface and signaling protein ycfS L,D-transpeptidaselinking Lpp to murein

TABLE 3 Gene Function of gene product lepB leader peptidase (signalpeptidase I) rnc RNase III era factor; Global regulatory functions dapAdihydrodipicolinate synthase gcvR DNA-binding transcriptional repressor,regulatory protein accessory to GcvA bcp peroxiredoxin; thiolperoxidase, thioredoxin-dependent hyfA hydrogenase 4, 4Fe—4S subunitrpoE RNA polymerase, sigma 24 (sigma E) factor nadB quinolinatesynthase, L-aspartate oxidase (B protein) subunit yfiC tRNA1(Val)(adenine(37)-N6)-methyltransferase srmB ATP-dependent RNA helicase g1414Putative transposase g1413 putative transposase nuoE NADH: ubiquinoneoxidoreductase, chain E nuoF NADH: ubiquinone oxidoreductase, chain FnuoG NADH: ubiquinone oxidoreductase, chain G glmZ sRNA antisenseactivator of glmS mRNA, Hfq-dependent hemY putative protoheme IXsynthesis protein hemX putative uroporphyrinogen III methyltransferasehemD uroporphyrinogen III synthase rlmL fused 23S rRNA m(2)G2445 andm(7)G2069 methyltransferase, SAM-dependent artQ arginine transportersubunit artM arginine transporter subunit artJ arginine binding protein,periplasmic rlmC 23S rRNA m(5)U747 methyltransferase, SAM-dependent ybjOInner membrane protein, DUF2593 family yejO autotransporter outermembrane homology yejM putative hydrolase, inner membrane yejL conservedprotein, UPF0352 family rpoS RNA polymerase, sigma S (sigma 38) factorygbN putative transporter ygbM hypothetical protein ygbL putative classII aldolase g3798 SOS-response transcriptional repressers (RecA-mediatedautopeptidases) g3797 hypothetical protein g3796 hypothetical proteing3795 hypothetical protein g3794 Superinfection exclusion protein Bg3793 Restriction inhibitor protein ral (Antirestriction protein) g3792hypothetical protein ryjA Novel sRNA, function unknown soxR DNA-bindingtranscriptional dual regulator, Fe—S center for redox-sensing soxSDNA-binding transcriptional dual regulator yjcC putativemembrane-anchored cyclic-di-GMP phosphodiesterase yjcB hypotheticalprotein efeU ferrous iron permease (pseudogene) efeO inactive ferrousion transporter EfeUOB

It is an aspect of the present invention to provide an Escherichiabacterium having a heparosan-producing ability, wherein:

the bacterium has been modified so that expression is increased of agene selected from the group consisting of rpoE, rbsR, rbsK, rbsB, hsrA,glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, rfaH, nusG,pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453,yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB,yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA,paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, lepB, rnc, era, dapA,gcvR, bcp, hyfA, nadB, yfiC, srmB, g1414, g1413, nuoE, nuoF, nuoG, glmZ,hemY, hemX, hemD, rlmL, artQ, artM, artJ, rlmC, ybjO, yejO, yejM, yejL,rpoS, ygbN, ygbM, ygbL, g3798, g3797, g3796, g3795, g3794, g3793, g3792,ryjA, soxR, soxS, yjcC, yjcB, efeU, efeO, and combinations thereof.

It is an aspect of the present invention to provide the the bacteriumbacterium as described above, which has been modified so that expressionof at least the rpoE gene is increased.

It is an aspect of the present invention to provide the the bacterium asdescribed above, which has been modified so that expression of at leastthe rfaH gene is increased.

It is an aspect of the present invention to provide the the bacterium asdescribed above, which has been further modified so that expression of agene selected from the group consisting of rbsR, rbsK, rbsB, hsrA, glgB,glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, nusG, pcoR, pcoS,pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB,mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD,thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC,gsiD, yliE, irp2, irp1, bhsA, ycfS, and combinations thereof.

It is an aspect of the present invention to provide the the bacterium asdescribed above, wherein said expression is increased by increasing thecopy number of the gene(s), and/or modifying a gene expression controlsequence of the gene(s).

It is an aspect of the present invention to provide the the bacterium asdescribed above, which is Escherichia coli.

It is an aspect of the present invention to provide the the bacterium asdescribed above, wherein:

the rbsB gene is a DNA comprising the nucleotide sequence of positions800 to 1690 of SEQ ID NO: 29, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 800 to 1690 of SEQ ID NO: 29 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rbsK gene is a DNA comprising the nucleotide sequence of positions1816 to 2745 of SEQ ID NO: 29, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1816 to 2745 of SEQ ID NO: 29 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rbsR gene is a DNA comprising the nucleotide sequence of positions2749 to 3741 of SEQ ID NO: 29, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 2749 to 3741 of SEQ ID NO: 29 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the hsrA gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 3707 to 5134 of SEQ ID NO: 29, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 3707to 5134 of SEQ ID NO: 29 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the glgB gene is a DNA comprising the nucleotide sequence of positions989 to 3175 of SEQ ID NO: 34, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 989 to 3175 of SEQ ID NO: 34 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the glgX gene is a DNA comprising the nucleotide sequence of positions3172 to 5145 of SEQ ID NO: 34, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 3172 to 5145 of SEQ ID NO: 34 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rcsB gene is a DNA comprising the nucleotide sequence of positions3312 to 3962 of SEQ ID NO: 43, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 3312 to 3962 of SEQ ID NO: 43 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rcsD gene is a DNA comprising the nucleotide sequence of positions623 to 3295 of SEQ ID NO: 43, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 623 to 3295 of SEQ ID NO: 43 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the micF gene is a DNA comprising the nucleotide sequence of positions219 to 311 of SEQ ID NO: 43, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 219 to 311 of SEQ ID NO: 43 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ybiX gene is a DNA comprising the nucleotide sequence of positions718 to 1395 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 718 to 1395 of SEQ ID NO: 37 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ybiI gene is a DNA comprising the nucleotide sequence of positions1469 to 1735 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1469 to 1735 of SEQ ID NO: 37 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ybiJ gene is a DNA comprising the nucleotide sequence of positions2000 to 2260 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 2000 to 2260 of SEQ ID NO: 37 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ybiC gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2488 to 3574 of SEQ ID NO: 37, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 2488to 3574 of SEQ ID NO: 37 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the ybiB gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 3715 to 4677 of SEQ ID NO: 37, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 3715to 4677 of SEQ ID NO: 37 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the rfaH gene is a DNA comprising the nucleotide sequence of SEQ ID NO:46, or a DNA comprising a nucleotide sequence having an identity of 90%or higher to the nucleotide sequence of SEQ ID NO: 46 and is able toincrease heparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the nusG gene is a DNA comprising the nucleotide sequence of SEQ ID NO:48, or a DNA comprising a nucleotide sequence having an identity of 90%or higher to the nucleotide sequence of SEQ ID NO: 48 and is able toincrease heparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the pcoR gene is a DNA comprising the nucleotide sequence of positions128 to 808 of SEQ ID NO: 50, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 128 to 808 of SEQ ID NO: 50 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the pcoS gene is a DNA comprising the nucleotide sequence of positions805 to 2205 of SEQ ID NO: 50, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 805 to 2205 of SEQ ID NO: 50 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the pcoE gene is a DNA comprising the nucleotide sequence of positions2423 to 2857 of SEQ ID NO: 50, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 2423 to 2857 of SEQ ID NO: 50 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yhcN gene is a DNA comprising the nucleotide sequence of positions63 to 326 of SEQ ID NO: 54, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 63 to 326 of SEQ ID NO: 54 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yhcO gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 382 to 654 of SEQ ID NO: 54, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 382to 654 of SEQ ID NO: 54 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the aaeB gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 746 to 2713 of SEQ ID NO: 54, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 746to 2713 of SEQ ID NO: 54 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the aaeA gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2719 to 3651 of SEQ ID NO: 54, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 2719to 3651 of SEQ ID NO: 54 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the aaeX gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 3659 to 3931 of SEQ ID NO: 54, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 3659to 3931 of SEQ ID NO: 54 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the g1455 gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 568 to 1140 of SEQ ID NO: 60, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 568to 1140 of SEQ ID NO: 60 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the alpA gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 1226 to 1486 of SEQ ID NO: 60, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 1226to 1486 of SEQ ID NO: 60 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the g1453 gene is a DNA comprising the nucleotide sequence of positions2389 to 2529 of SEQ ID NO: 60, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 2389 to 2529 of SEQ ID NO: 60 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yrbA gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 977 to 1246 of SEQ ID NO: 64, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 977to 1246 of SEQ ID NO: 64 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the mlaB gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 1391 to 1780 of SEQ ID NO: 64, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 1391to 1780 of SEQ ID NO: 64 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the mlaC gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 1684 to 2319 of SEQ ID NO: 64, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 1684to 2319 of SEQ ID NO: 64 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the mlaD gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2338 to 2889 of SEQ ID NO: 64, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 2338to 2889 of SEQ ID NO: 64 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the mlaE gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2894 to 3676 of SEQ ID NO: 64, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 2894to 3676 of SEQ ID NO: 64 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the mlaF gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 3684 to 4493 of SEQ ID NO: 64, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 3684to 4493 of SEQ ID NO: 64 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the yrbG gene is a DNA comprising the nucleotide sequence of positions4703 to 5680 of SEQ ID NO: 64, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 4703 to 5680 of SEQ ID NO: 64 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the norW gene is a DNA comprising the nucleotide sequence of positions1201 to 2334 of SEQ ID NO: 72, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1201 to 2334 of SEQ ID NO: 72 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ybjI gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 117 to 932 of SEQ ID NO: 74, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 117to 932 of SEQ ID NO: 74 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the ybjJ gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 932 to 2140 of SEQ ID NO: 74, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 932to 2140 of SEQ ID NO: 74 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the ybjK gene is a DNA comprising the nucleotide sequence of positions2224 to 2760 of SEQ ID NO: 74, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 2224 to 2760 of SEQ ID NO: 74 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rybB gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2777 to 2855 of SEQ ID NO: 74, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 2777to 2855 of SEQ ID NO: 74 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the yjjY gene is a DNA comprising the nucleotide sequence of positions124 to 264 of SEQ ID NO: 78, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 124 to 264 of SEQ ID NO: 78 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yjtD gene is a DNA comprising the nucleotide sequence of positions664 to 1350 of SEQ ID NO: 78, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 664 to 1350 of SEQ ID NO: 78 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the thrL gene is a DNA comprising the nucleotide sequence of positions1564 to 1629 of SEQ ID NO: 78, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1564 to 1629 of SEQ ID NO: 78 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the thrA gene is a DNA comprising the nucleotide sequence of positions1711 to 4173 of SEQ ID NO: 78, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1711 to 4173 of SEQ ID NO: 78 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the thrB gene is a DNA comprising the nucleotide sequence of positions4175 to 5107 of SEQ ID NO: 78, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 4175 to 5107 of SEQ ID NO: 78 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the fruA gene is a DNA comprising the nucleotide sequence of positions897 to 2588 of SEQ ID NO: 84, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 897 to 2588 of SEQ ID NO: 84 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the psuK gene is a DNA comprising the nucleotide sequence of positions3165 to 3953 of SEQ ID NO: 84, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 3165 to 3953 of SEQ ID NO: 84 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ytfT gene is a DNA comprising the nucleotide sequence of positions252 to 1277 of SEQ ID NO: 87, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 252 to 1277 of SEQ ID NO: 87 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yjfF gene is a DNA comprising the nucleotide sequence of positions1264 to 2259 of SEQ ID NO: 87, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1264 to 2259 of SEQ ID NO: 87 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the fbp gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2292 to 3290 of SEQ ID NO: 87, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 2292to 3290 of SEQ ID NO: 87 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the yagU gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 117 to 731 of SEQ ID NO: 91, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 117to 731 of SEQ ID NO: 91 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the paoA gene is a DNA comprising the nucleotide sequence of positions1149 to 1838 of SEQ ID NO: 91, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1149 to 1838 of SEQ ID NO: 91 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the paoB gene is a DNA comprising the nucleotide sequence of positions1835 to 2791 of SEQ ID NO: 91, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1835 to 2791 of SEQ ID NO: 91 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the gsiC gene is a DNA comprising the nucleotide sequence of positions264 to 1184 of SEQ ID NO: 95, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 264 to 1184 of SEQ ID NO: 95 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the gsiD gene is a DNA comprising the nucleotide sequence of positions1187 to 2098 of SEQ ID NO: 95, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 1187 to 2098 of SEQ ID NO: 95 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yliE gene is a DNA comprising the nucleotide sequence of positions2276 to 4624 of SEQ ID NO: 95, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 2276 to 4624 of SEQ ID NO: 95 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the irp2 gene is a DNA comprising the nucleotide sequence of SEQ ID NO:100, or a DNA comprising a nucleotide sequence having an identity of 90%or higher to the nucleotide sequence of SEQ ID NO: 100 and is able toincrease heparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the irp1 gene is a DNA comprising the nucleotide sequence of SEQ ID NO:102, or a DNA comprising a nucleotide sequence having an identity of 90%or higher to the nucleotide sequence of SEQ ID NO: 102 and is able toincrease heparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the bhsA gene is a DNA comprising the nucleotide sequence of positions440 to 697 of SEQ ID NO: 104, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 440 to 697 of SEQ ID NO: 104 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ycfS gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 779 to 1741 of SEQ ID NO: 104, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 779to 1741 of SEQ ID NO: 104 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the lepB gene is a DNA comprising the nucleotide sequence of positions1344 to 2318 of SEQ ID NO: 107, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1344 to 2318 of SEQ ID NO: 107 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rnc gene is a DNA comprising the nucleotide sequence of positions2590 to 3270 of SEQ ID NO: 107, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 2590 to 3270 of SEQ ID NO: 107 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the era gene is a DNA comprising the nucleotide sequence of positions3267 to 4172 of SEQ ID NO: 107, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 3267 to 4172 of SEQ ID NO: 107 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the dapA gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 858 to 1736 of SEQ ID NO: 111, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 858to 1736 of SEQ ID NO: 111 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the gcvR gene is a DNA comprising the nucleotide sequence of positions1882 to 2454 of SEQ ID NO: 111, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1882 to 2454 of SEQ ID NO: 111 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the bcp gene is a DNA comprising the nucleotide sequence of positions2454 to 2924 of SEQ ID NO: 111, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 2454 to 2924 of SEQ ID NO: 111 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the hyfA gene is a DNA comprising the nucleotide sequence of positions3177 to 3794 of SEQ ID NO: 111, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 3177 to 3794 of SEQ ID NO: 111 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rpoE gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 355 to 930 of SEQ ID NO: 116, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 355to 930 of SEQ ID NO: 116 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the nadB gene is a DNA comprising the nucleotide sequence of positions1338 to 2960 of SEQ ID NO: 116, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1338 to 2960 of SEQ ID NO: 116 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yfiC gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2945 to 3682 of SEQ ID NO: 116, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions2945 to 3682 of SEQ ID NO: 116 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the srmB gene is a DNA comprising the nucleotide sequence of positions3814 to 5148 of SEQ ID NO: 116, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 3814 to 5148 of SEQ ID NO: 116 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g1414 gene is a DNA comprising the nucleotide sequence of positions28 to 699 of SEQ ID NO: 121, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 28 to 699 of SEQ ID NO: 121 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g1413 gene is a DNA comprising the nucleotide sequence of positions831 to 1157 of SEQ ID NO: 121, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 831 to 1157 of SEQ ID NO: 121 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the nuoE gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 796 to 1296 of SEQ ID NO: 124, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 796to 1296 of SEQ ID NO: 124 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the nuoF gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 1293 to 2630 of SEQ ID NO: 124, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions1293 to 2630 of SEQ ID NO: 124 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the nuoG gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2683 to 5409 of SEQ ID NO: 124, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions2683 to 5409 of SEQ ID NO: 124 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the glmZ gene is a DNA comprising the nucleotide sequence of positions357 to 563 of SEQ ID NO: 128, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 357 to 563 of SEQ ID NO: 128 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the hemY gene is a DNA comprising the nucleotide sequence of positions611 to 1807 of SEQ ID NO: 128, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 611 to 1807 of SEQ ID NO: 128 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the hemX gene is a DNA comprising the nucleotide sequence of positions1810 to 2991 of SEQ ID NO: 128, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1810 to 2991 of SEQ ID NO: 128 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the hemD gene is a DNA comprising the nucleotide sequence of positions3013 to 3753 of SEQ ID NO: 128, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 3013 to 3753 of SEQ ID NO: 128 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rlmL gene is a DNA comprising the nucleotide sequence of positions571 to 2679 of SEQ ID NO: 132, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 571 to 2679 of SEQ ID NO: 132 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the artQ gene is a DNA comprising the nucleotide sequence of positions386 to 1102 of SEQ ID NO: 134, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 386 to 1102 of SEQ ID NO: 134 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the artM gene is a DNA comprising the nucleotide sequence of positions1102 to 1770 of SEQ ID NO: 134, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1102 to 1770 of SEQ ID NO: 134 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the artJ gene is a DNA comprising the nucleotide sequence of positions2061 to 2792 of SEQ ID NO: 134, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 2061 to 2792 of SEQ ID NO: 134 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rlmC gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2991 to 4118 of SEQ ID NO: 134, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions2991 to 4118 of SEQ ID NO: 134 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ybjO gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 4159 to 4647 of SEQ ID NO: 134, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions4159 to 4647 of SEQ ID NO: 134 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yejO gene is a DNA comprising the nucleotide sequence of positions216 to 2807 of SEQ ID NO: 140, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 216 to 2807 of SEQ ID NO: 140 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yejM gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 3061 to 4821 of SEQ ID NO: 140, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions3061 to 4821 of SEQ ID NO: 140 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yejL gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 4841 to 5068 of SEQ ID NO: 140, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions4841 to 5068 of SEQ ID NO: 140 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the rpoS gene is a DNA comprising the nucleotide sequence of positions318 to 1310 of SEQ ID NO: 144, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 318 to 1310 of SEQ ID NO: 144 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ygbN gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 1404 to 2768 of SEQ ID NO: 144, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions1404 to 2768 of SEQ ID NO: 144 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ygbM gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 2857 to 3633 of SEQ ID NO: 144, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions2857 to 3633 of SEQ ID NO: 144 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ygbL gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 3638 to 4276 of SEQ ID NO: 144, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions3638 to 4276 of SEQ ID NO: 144 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g3798 gene is a DNA comprising the nucleotide sequence of positions615 to 1268 of SEQ ID NO: 149, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 615 to 1268 of SEQ ID NO: 149 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g3797 gene is a DNA comprising the nucleotide sequence of positions1368 to 2219 of SEQ ID NO: 149, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1368 to 2219 of SEQ ID NO: 149 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g3796 gene is a DNA comprising the nucleotide sequence of positions2257 to 2748 of SEQ ID NO: 149, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 2257 to 2748 of SEQ ID NO: 149 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g3795 gene is a DNA comprising the nucleotide sequence of positions3021 to 3203 of SEQ ID NO: 149, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 3021 to 3203 of SEQ ID NO: 149 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g3794 gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 3470 to 4051 of SEQ ID NO: 149, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions3470 to 4051 of SEQ ID NO: 149 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g3793 gene is a DNA comprising the nucleotide sequence of positions4280 to 4480 of SEQ ID NO: 149, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 4280 to 4480 of SEQ ID NO: 149 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the g3792 gene is a DNA comprising the nucleotide sequence of positions4520 to 4717 of SEQ ID NO: 149, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 4520 to 4717 of SEQ ID NO: 149 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the ryjA gene is a DNA comprising the nucleotide sequence of positions657 to 796 of SEQ ID NO: 157, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 657 to 796 of SEQ ID NO: 157 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the soxR gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 790 to 1254 of SEQ ID NO: 157, or a DNAcomprising a nucleotide sequence having an identity of 90% or higher tothe complementary sequence of the nucleotide sequence of positions 790to 1254 of SEQ ID NO: 157 and is able to increase heparosan-producingability of an Escherichia bacterium having heparosan-producing abilitywhen expression amount thereof is increased in the bacterium;

the soxS gene is a DNA comprising the nucleotide sequence of positions1340 to 1663 of SEQ ID NO: 157, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1340 to 1663 of SEQ ID NO: 157 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yjcC gene is a DNA comprising the complementary sequence of thenucleotide sequence of positions 1666 to 3252 of SEQ ID NO: 157, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the complementary sequence of the nucleotide sequence of positions1666 to 3252 of SEQ ID NO: 157 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the yjcB gene is a DNA comprising the nucleotide sequence of positions3682 to 3963 of SEQ ID NO: 157, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 3682 to 3963 of SEQ ID NO: 157 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium;

the efeU gene is a DNA comprising the nucleotide sequence of positions753 to 1583 of SEQ ID NO: 162, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence ofpositions 753 to 1583 of SEQ ID NO: 162 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium; and

the efeO gene is a DNA comprising the nucleotide sequence of positions1641 to 2768 of SEQ ID NO: 162, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the nucleotide sequenceof positions 1641 to 2768 of SEQ ID NO: 162 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium.

It is a further aspect of the present invention to provide a method forproducing heparosan, the method comprising:

culturing the bacterium as described above in a medium to produce andaccumulate heparosan in the medium; and

collecting the heparosan from the medium.

It is an aspect of the present invention to provide the method forproducing heparin, the method comprising:

culturing the bacterium mentioned above in a medium to produce andaccumulate heparosan in the medium;

chemically and/or enzymatically treating the heparosan to produceheparin; and

collecting the heparin.

The functions of the gene products of the genes mentioned in Tables 1 to3, and relations thereof with heparosan production will be describedbelow.

RbsR, RbsK, and RbsB are factors that participate in incorporation anduse of D-ribose. RbsR is a repressor of ribose metabolism, andnegatively controls transcription of the rbs operon encoding proteinsthat participate in a catabolic reaction of ribose (Laikova O. N. et al.(2001) “Computational analysis of the transcriptional regulation ofpentose utilization systems in the gamma subdivision of Proteobacteria”,FEMS Microbiol Lett., 205(2):315-22). RbsK is a ribokinase, andcatalyzes phosphorylation of D-ribose (Bork P. et al. (1993) “Convergentevolution of similar enzymatic function on different protein folds: thehexokinase, ribokinase, and galactokinase families of sugar kinases”,Protein Sci., 2(1):31-40). RbsB is one of the subunits of the ribose ABCtransporter, and the ribose ABC transporter carries out incorporation ofD-ribose (Iida A. et al. (1984) “Molecular cloning and characterizationof genes required for ribose transport and utilization in Escherichiacoli K-12”, J. Bacteriol., 158(2):674-82). There has not been any reportindicating the relationship between these proteins and heparosanproduction.

HsrA is an inner membrane protein presumed to be a member of the majorfacilitator superfamily (WS) (Pao S. S. et al. (1998) “Major facilitatorsuperfamily”, Microbiol. Mol. Biol. Rev., 62(1):1-34). HsrA is presumedto have a function of a proton-driven type drug efflux system on thebasis of sequence homology, but the actual function thereof has not beenidentified. Therefore, there has not been any report indicating therelationship between this protein and heparosan production.

GlgB and GlgX are enzymes that participate in the biosynthesis anddecomposition of glycogen, respectively. GlgB is a glycogen-branchingenzyme (1,4-α-glucan-branching enzyme), and introduces branching into apolyglucose chain by forming α-1,6-glycosidic linkage during theglycogen biosynthesis process (Boyer C. and Preiss (1977) “Biosynthesisof bacterial glycogen: Purification and properties of the Escherichiacoli b alpha-1,4,-glucan: alpha-1,4-glucan 6-glycosyltansferase”, J.Biochemistry, 16(16):3693-9). GlgX is a glycogen-debranching enzyme, andit hydrolyzes α-1,6-glycosidic linkage to liberate a unit of 3 or 4glucose residues, and thereby eliminates branching of glycogen(Dauvillee D. et al. (2005) “Role of the Escherichia coli glgX gene inglycogen metabolism”, J. Bacteriol., 187(4):1465-73). There has not beenany report indicating the relationship between these proteins andheparosan production.

It is known that micF is an antisense RNA that participates in theexpression suppression of OmpF, and functions especially under anosmotic pressure condition (Ramani N. (1994) “micF antisense RNA has amajor role in osmoregulation of OmpF in Escherichia coli”, J.Bacteriol., 176:5005-5010). There has not been any report indicating therelationship between this nucleotide chain and heparosan production.

RcsB is a transcription control factor found in bacteria belonging tothe genus Escherichia, Salmonella, Klebsiella, or the like, and it isconsidered to control mainly the biosynthesis of cholanic acid, which isa capsule constituent component (Majdalani N. et al. (2005) “The Rcsphosphorelay: a complex signal transduction system”, Anuu. Rev.Microbiol., 59:379-405). It has been reported that RcsB participates inthe Vi polysaccharide expression of Citrobacter freundii (Houng H. S. etal. (1992) “Expression of Vi antigen in Escherichia coli K-12:characterization of ViaB from Citrobacter freundii and identity of ViaAwith RcsB”, J. Bacteriol., 174:5910-5915), and expression of K2 capsuleof Klebsiella bacteria (Rochaporn W. et al. (1992) “Involvement of rcsBin Klebsiella K2 Capsule Synthesis in Escherichia coli K-12”, J.Bacteriol. 174:1063-1067). Moreover, it is known that overexpression ofRcsB increases K30 capsular polysaccharide production, but it has beenreported that RcsB does not participate in the transcription of the cspcluster encoding polymerization enzymes for the K30 capsularpolysaccharide, but positively controls expression of the galF geneencoding a biosynthesis enzyme of UDP-glucose, which is a precursor ofthe polysaccharide (Andrea Rahn et al. (2003) “Transcriptionalorganization and regulation of The Escherichia coli”, Mol. Microbiol.,47:1045-1060). It has also been reported that overexpression of RcsBdoes not increase production of K5 capsular polysaccharide (heparosan)or K1 capsular polysaccharide (Wendy J. Keenleyside et al. (1993)“Coexpression of Colanic Acid and Serotype-Specific CapsularPolysaccharides in Escherichia coli Strains with Group II K Antigens”,J. Bacteriol., 175:6725-6730). RcsD is a sensor protein having histidinekinase, and it is known to transfer a phosphate group to RcsB inresponse to an external stimulus.

YbiX, Ybil, YbiJ, YbiC, and YbiB are factors with unknown function.Therefore, there has not been any report indicating the relationshipbetween these proteins and heparosan production.

RfaH is a transcription factor required for the biosynthesis oflipopolysaccharides, secretion of α-hemolysin, and production of the Ffactor in Escherichia coli and Salmonella typhimurium (Leeds J. A. andWelch R. A. (1996) “RfaH enhances elongation of Escherichia coli hlyCABDmRNA”, J. Bacteriol., 178(7):1850-7). It is also known that, in theEscherichia coli K5 strain, RfaH is required for the K5 capsularformation (Stevens M. P. et al. (1994) “Regulation of Escherichia coliK5 capsular polysaccharide expression: Evidence for involvement of RfaHin the expression of group II capsules”, FEMS Microbiol. Lett.,124(1):93-98), and RfaH binds to the promoter region of the region 3(kpsM, kpsT) to positively control transcription of not only the region3, but also the downstream region 2 (kfiA, kfiB, kfiC, and kfiD) (Xue P.et al. (2009) “Regulation of expression of the region 3 promoter of theEscherichia coli K5 capsule gene cluster involves H-NS, SlyA, and alarge 5′ untranslated region”, J. Bacteriol., 191(6):1838-1846).However, influence of enhanced expression of the rfaH gene on the amountof heparosan production has not been examined in the Escherichia coli K5strain nor any other heparosan-producing bacteria.

NusG is a transcription factor, and is considered to regulatetranscription by interacting with RNA polymerase (Li J. et al. (1992) J.Biol. Chem., 267(9):6012-6019). It is also reported that NusGparticipates in the capsule biosynthesis of Bacteroides fragilis(Livanis M. et al. (2009) J. Bacteriol., 191(23):7288-7295). However,there has not so far been reported involvement thereof in the heparosanbiosynthesis. It is considered that NusG is a homologue of RfaH, andNusG and RfaH have a common domain (Bailey M. et al. (1996) Mol.Microbiol., 22(4):7729-737). However, the amino acid sequence homologyof NusG and RfaH is about 20% in all of the Escherichia coli K-12strains, K5 strains, and B strains, and it cannot be said that theseproteins are highly homologous.

PcoR, PcoS, and PcoE are factors that relate to copper resistance. PcoRand PcoS are highly homologous to the activator of the pco operon, andthe sensor protein of the two-component control system that responds toenvironmental stimuli, respectively (Cooksey D. A. (2006) “Copper uptakeand resistance in bacteria”, Mol. Microbiol., 7(1):1-5). PcoE is acopper-binding protein. There has not been any report indicating therelationship between these proteins and heparosan production.

YhcN is a factor involved in response of bacterial cells to hydrogenperoxide stress. A yhcN gene-deficient strain shows improved sensitivityto hydrogen peroxide, and increased biofilm formation amount (Lee J. etal. (2010) “Identification of stress-related proteins in Escherichiacoli using the pollutant cis-dichloroethylene”, J. Appl. Microbiol.,June; 108(6):2088-102). There has not been any report indicating therelationship between this protein and heparosan production.

YhcO shows homology to an inhibition factor for barnase, which is atoxic RNase derived from Bacillus amyloliquefaciens. However,Escherichia bacteria do not have an RNase of the barnase family, and thefunction of YhcO is unclear. Therefore, there has not been any reportindicating the relationship between this protein and heparosanproduction.

AaeB and AaeA are subunits of an efflux carrier of 4-hydroxybenzoicacid. Although AaeX is also estimated to be an efflux carrier, actualfunction thereof is unknown (Van Dyk T. K. et al. (2004)“Characterization of the Escherichia coli AaeAB efflux pump: a metabolicrelief valve?”, J. Bacteriol., 186:7196-7204). There has not been anyreport indicating the relationship between these proteins and heparosanproduction.

The g1455 and g1453 genes are found in only the Escherichia coli K5strain, and the functions of the proteins encoded by these genes areunknown. Therefore, there has not been any report indicating therelationship between these proteins and heparosan production.

AlpA is an expression regulator of the intA gene encoding a prophageintegrase, and it has a function of complementing deficiency of the Lonprotease through increase of expression of intA (Trempy J. E. et al.(1994) “Alp suppression of Lon: dependence on the slpA gene”, J.Bacteriol., 176(7):2061-7). Although AlpA may possibly relate to biofilmformation or capsule production (Herzberg M. et al. (2006) “YdgG (TqsA)controls biofilm formation in Escherichia coli K-12 through autoinducer2 transport”, J. Bacteriol., 188(2):587-98), there has not been anyreport indicating the relationship between AlpA and heparosanproduction.

YrbA (synonym is IbaG) is a factor presumed to be a DNA-binding typetranscription factor, and expression amount thereof increases underacidity stress conditions (Guinote I. B. et al. (2012) “Characterizationof the BolA homolog IbaG: a new gene involved in acid resistance”, J.Microbiol. Biotechnol., 22(4):484-93). There has not been any reportindicating the relationship between this protein and heparosanproduction.

MlaB, MlaC, MlaD, MlaE, and MlaF are constituent factors of aphospholipid ABC transporter, and participate in transportation ofphospholipids and maintenance of lipid asymmetry (Malinverni J. C. andSilhavy T. J. (2009) “An ABC transport system that maintains lipidasymmetry in the gram-negative outer membrane”, Proc. Natl. Acad. Sci.USA, 106(19):8009-14). There has not been any report indicating therelationship between these proteins and heparosan production.

YrbG is a 5-pass transmembrane type inner membrane protein, and it ispresumed to be a Na⁺/Ca²⁺ antiporter on the basis of sequence homology.However, intracellular Ca²⁺ level regulation ability of YrbG has notbeen confirmed, and actual function thereof is unknown (Naseem R. et al.(2008) “pH and monovalent cations regulate cytosolic free Ca(2+) in E.coli”, Biochim. Biophys. Acta, 1778(6):1415-22). Therefore, there hasnot been any report indicating the relationship between this protein andheparosan production at all, either.

NorW is a nitric oxide (NO) reductase to be expressed in response to NOstress (Gardner A. M. et al. (2003) “Role of NorR and sigma54 in thenitric oxide stress response”, J. Biol. Chem., 278(12):10081-6). Therehas not been any report indicating the relationship between this proteinand heparosan production.

YbjI is a flavin mononucleotide (FMN) phosphorylation enzyme belongingto the haloacid dehalogenation enzyme-like hydrolase family (KuznetsovaE. et al. (2006) “Genome-wide analysis of substrate specificities of theEscherichia coli haloacid dehalogenase-like phosphatase family”, J.Biol. Chem., 281(47):36149-61). There has not been any report indicatingthe relationship between this protein and heparosan production.

YbjJ and YbjK are proteins with unknown function. Therefore, there hasnot been any report indicating the relationship between these proteinsand heparosan production, either.

RybB is a low molecular weight RNA, the expression of which is dependenton sigma factor GE, which is activated in response to cell surfacestress, and suppresses synthesis of the sigma factor GE (Thompson K. M.et al. (2007) “SigmaE regulates and is regulated by a small RNA inEscherichia coli”, J. Bacteriol., 189(11):4243-56). RybB alsoparticipates in expression inhibition of OmpC and OmpW (Johansen J. etal. (2006) “Conserved small non-coding RNAs that belong to the sigmaEregulon: role in down-regulation of outer membrane proteins”, J. Mol.Biol., 364(1):1-8). There has not been any report indicating therelationship between RybB and heparosan production.

YjjY is a protein of unknown function. Therefore, there has not been anyreport indicating the relationship between this protein and heparosanproduction at all.

Although YjtD is presumed to be one of RNA methyltransferases, itsactual function is unknown (Anantharaman V. et al. (2002) “SPOUT: aclass of methyltransferases that includes spoU and trmD RNA methylasesuperfamilies, and novel superfamilies of predicted prokaryotic RNAmethylases”, J. Mol. Microbiol. Biotechnol., 4(1):71-5). Therefore,there has not been any report indicating the relationship between thisprotein and heparosan production.

ThrB, ThrA, and ThrL are enzymes of the threonine biosynthesis pathway.ThrB is a homoserine kinase that catalyzes the reaction of convertinghomoserine into O-phospho-L-homoserine, and participates in thebiosynthesis of threonine (Burr B. et al. (1976) “Homoserine kinase fromEscherichia coli K12”, Eur. J. Biochem., 62(3):519-26). ThrA is anenzyme having the dual functions of aspartate kinase I and homoserinedehydrogenase I, and participates in the biosyntheses of lysine andmethionine, in addition to that of threonine (Clark R. B., Ogilvie J. W.et al. (1972) “Aspartokinase I-homoserine dehydrogenase I of Escherichiacoli K12: Subunit molecular weight and nicotinamide-adenine dinucleotidephosphate binding”, Biochemistry, 11(7):1278-82). ThrL is a leaderpeptide of the thrLABC operon, and attenuates expression of the thrLABCoperon depending on the concentrations of threonine and isoleucine (LynnS. P. et al. (1982) “Attenuation regulation in the thr operon ofEscherichia coli K-12: molecular cloning and transcription of thecontrolling region”, J. Bacteriol., 152(1):363-71). There has not beenany report indicating the relationship between these proteins andheparosan production.

FruA is a fructose PTS permease, and has the IIB domain and IIC domain(Prior T. I. and Kornberg H. L. (1988) “Nucleotide sequence of fruA, thegene specifying enzyme IIfru of the phosphoenolpyruvate-dependent sugarphosphotransferase system in Escherichia coli K12”, J. Gen. Microbiol.,134(10):2757-68). There has not been any report indicating therelationship between this protein and heparosan production.

PsuK is a pseudouridine kinase and participates in catabolism ofpseudouridine, which is a modified RNA frequently found in the PIT loopof tRNA (Solomon L. R. and Breitman T. R. (1971) “Pseudouridine kinaseof Escherichia coli: a new enzyme”, Biochem. Biophys. Res. Commun.,44(2):299-304). There has not been any report indicating therelationship between this protein and heparosan production.

Although YtfT and YjfF are presumed to be membrane constituentcomponents of the galactose ABC carrier, their actual function isunknown. Therefore, there has not been any report indicating therelationship between these proteins and heparosan production at all,either.

Fbp is a fructose-1,6-diphosphate phosphatase(fructose-1,6-bisphosphatase) that catalyzes the reaction of convertingfructose-1,6-diphosphate into fructose-6-phosphate in thegluconeogenesis pathway (Fraenkel D. G and Horecker B. L. (1965)“Fructose-1,6-diphosphatase and acid hexose phosphatase of Escherichiacoli”, J. Bacteriol., 90(4):837-42). There has not been any reportindicating the relationship between this protein and heparosanproduction.

Although YagU is presumed to be an inner membrane protein, its functionis unknown. Therefore, there has not been any report indicating therelationship between this protein and heparosan production, either.

PaoA (also called YagT) and PaoB (also called YagS) are constituentfactors of the aldehyde oxidoreductase YagTSR. PaoA is an iron-bindingsubunit, and PaoB is a flavine-adenine dinucleotide (FAD)-bindingsubunit (Neumann M. et al. (2009) “A periplasmic aldehyde oxidoreductaserepresents the first molybdopterin cytosine dinucleotide cofactorcontaining molybdo-flavoenzyme from Escherichia coli”, FEBS J.,276(10):2762-74). There has not been any report indicating therelationship between these proteins and heparosan production.

GsiC and GsiD are constituent factors of a glutathione ABC transportcarrier. GsiC and GsiD localize in the inner membrane (Moussatova A. etal. (2008) “ATP-binding cassette transporters in Escherichia coli”,Biochim. Biophys. Acta, 1778(9):1757-71). There has not been any reportindicating the relationship between these proteins and heparosanproduction.

YliE is presumed to be a c-di-GMP-specific phosphodiesterase, andoverexpression thereof promotes biofilm formation (Boehm A. et al.(2009) “Second messenger signalling governs Escherichia coli biofilminduction upon ribosomal stress”, Mol. Microbiol., 72(6):1500-16). Therehas not been any report indicating the relationship between this proteinand heparosan production.

Irp2 and Irp1 are non-ribosomal peptide synthases, and participate iniron incorporation (Pelludat C. et al. (1998) “The yersiniabactinbiosynthetic gene cluster of Yersinia enterocolitica: organization andsiderophore-dependent regulation”, J. Bacteriol., 180(3):538-46). Therehas not been any report indicating the relationship between theseproteins and heparosan production.

BhsA (synonym is YcfR) is presumed to be an outer membrane protein, andparticipates in biofilm formation and stress response (Zhang X. S. etal. (2007) “YcfR (BhsA) influences Escherichia coli biofilm formationthrough stress response and surface hydrophobicity”, J. Bacteriol.,189(8):3051-62). There has not been any report indicating therelationship between this protein and heparosan production.

YcfS is one of L,D-transpeptidases. YcfS catalyzes the reaction ofremoving a D-alanine residue from the meso-diaminopimelate (DAP) residueof peptidoglycan, and binding a lysine residue of the C-terminus ofBraun lipoprotein to this meso-DAP residue. Through this reaction, thepeptidoglycan covalently binds to the outer membrane via the Braunlipoprotein (Magnet S. et al. (2007) “Identification of theL,D-transpeptidases responsible for attachment of the Braun lipoproteinto Escherichia coli peptidoglycan”, J. Bacteriol., 189(10):3927-31).There has not been any report indicating the relationship between thisprotein and heparosan production.

LepB is a signal peptidase that removes an N-terminus leader peptidefrom a secretory protein (Dalbey R. E. (1991) “Leader peptidase”, Mol.Microbiol., 5(12):2855-60). There has not been any report indicating therelationship between this protein and heparosan production.

Rnc is an RNaseIII that cleaves double stranded RNA to produce 5′phosphate group and hydroxyl group, and is required for processing ofrRNA or phage mRNA. The main roles of Rnc are regulation of geneexpression, and functionalization of antisense RNA (Robertson H. D. andDunn J. J. (1975) “Ribonucleic acid processing activity of Escherichiacoli ribonuclease III”, J. Biol. Chem., 25; 250(8):3050-6). There hasnot been any report indicating the relationship between this protein andheparosan production.

Era is a factor indispensable for survival (Takiff H. E. et al. (1992)“Locating essential Escherichia coli genes by using mini-Tn10transposons: the pdxJ operon”, J. Bacteriol., 174(5):1544-53). It hasbeen elucidated by the yeast two-hybrid method that Era interacts withMazG (Zhang J. and Inouye M., (2002) “MazG, a nucleoside triphosphatepyrophosphohydrolase, interacts with Era, an essential GTPase inEscherichia coli”, J. Bacteriol., 184 (19):5323-9). There has not beenany report indicating the relationship between this protein andheparosan production.

DapA is a 4-hydroxy-tetrahydrodipicolinate synthase.4-Hydroxy-tetrahydrodipicolinate synthase is one of the lysinebiosynthesis enzymes, and catalyzes the reaction of converting pyruvicacid and L-aspartic acid β-semialdehyde into(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate. This reaction isconsidered to be a rate-limiting step of the lysine biosynthesis afterthe reaction with aspartate kinase III (Laber B. et al. (1992)“Escherichia coli dihydrodipicolinate synthase: Identification of theactive site and crystallization”, Biochem. J., 288(Pt 2):691-5). Therehas not been any report indicating the relationship between this proteinand heparosan production.

GcvR is a protein presumed to be a transcriptional control factor, andparticipates in expression of glycine biosynthesis genes. In the absenceof glycine, GcvR directly binds to GcvA to form a GlvR/GlvA complex, andinhibits expression of glycine decomposition genes. In the presence ofglycine, glycine binds to GcvR to inhibit the formation of the GlvR/GlvAcomplex (Ghrist A. C. et al. (2001) “GcvR interacts with GcvA to inhibitactivation of the Escherichia coli glycine cleavage operon”,Microbiology, 147(Pt 8):2215-21). There has not been any reportindicating the relationship between this protein and heparosanproduction.

Bcp is a thioredoxin 1-dependent thiol peroxidase (Clarke D. J. et al.(2009) “Interrogating the molecular details of the peroxiredoxinactivity of the Escherichia coli bacterioferritin comigratory proteinusing high-resolution mass spectrometry”, Biochemistry, 48(18):3904-14).There has not been any report indicating the relationship between thisprotein and heparosan production.

HyfA has four 4Fe-4S clusters, and is presumed to participate inelectron transportation (Andrews S. C. et al. (1997) “A 12-cistronEscherichia coli operon (hyf) encoding a putative proton-translocatingformate hydrogenlyase system”, Microbiology, 143(Pt 11):3633-47). Therehas not been any report indicating the relationship between this proteinand heparosan production.

RpoE is sigma E (σ^(E)), which is one of the sigma factors and functionsas a subunit of RNA polymerase. RpoE controls expression of proteaseagainst membrane and intermembrane proteins in response to heat shockand stress (Ades S. E. et al. (2003) “Regulation of the alternativesigma factor sigma(E) during initiation, adaptation, and shutoff of theextracytoplasmic heat shock response in Escherichia coli”, J.Bacteriol., 185(8):2512-9). There has not been any report indicating therelationship between this protein and heparosan production.

NadB is an L-aspartate oxidase. L-Aspartate oxidase is an initiationenzyme of the de novo NAD biosynthesis pathway, and catalyzes thereaction of converting L-aspartic acid to iminoaspartic acid in aFAD-dependent manner (Mortarino M. et al. (1996) “L-aspartate oxidasefrom Escherichia coli, I. Characterization of coenzyme binding andproduct inhibition”, Eur. J. Biochem., 239(2):418-26). There has notbeen any report indicating the relationship between this protein andheparosan production.

YfiC is a methyltransferase that methylates N at the position 6 of A37(adenine at the position 37) of valine tRNA (Golovina A. Y. et al.(2009) RNA. “The yfiC gene of E. coli encodes an adenine-N6methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)”,15(6):1134-41). The nucleotide of the position 37 of tRNA is adjacent tothe anticodon triplet, and is often modified. There has not been anyreport indicating the relationship between this protein and heparosanproduction.

SrmB is a DEAD-box type RNA helicase that promotes reactions of an earlystage of 50S subunit assembly of ribosome (Charollais J. et al. (2003)“The DEAD-box RNA helicase SrmB is involved in the assembly of 50Sribosomal subunits in Escherichia coli”, Mol. Microbiol.,48(5):1253-65). There has not been any report indicating therelationship between this protein and heparosan production.

G1414 and G1413 are proteins of unknown functions. Therefore, there hasnot been any report indicating the relationship between these proteinsand heparosan production.

NuoE, NuoF, and NuoG are soluble fragments of an NADH dehydrogenase I,and function as the entrance of electrons into the electron transportsystem (Braun M. et al. (1998) “Characterization of the overproducedNADH dehydrogenase fragment of the NADH:ubiquinone oxidoreductase(complex I) from Escherichia coli”, Biochemistry., 37(7):1861-7). Therehas not been any report indicating the relationship between theseproteins and heparosan production.

GlmZ is a low molecular weight RNA that controls expression andtranslation of glmS mRNA by posttranscriptional modification in responseto the intracellular concentration of glucosamine-6-phosphate (KalamorzF. et al. (2007) “Feedback control of glucosamine-6-phosphate synthaseGlmS expression depends on the small RNA GlmZ and involves the novelprotein YhbJ in Escherichia coli”, Mol. Microbiol., 65(6):1518-33). GlmZdirectly binds to 5′-UTR of glmS mRNA to liberate the SD region of glmSmRNA, which had formed a loop structure, and thereby activatetranslation of glmS mRNA (Urban J. H. and Vogel J. et al. (2008) “Twoseemingly homologous noncoding RNAs act hierarchically to activate glmSmRNA translation”, PLoS Biol., 6(3):e64). GlmS isL-glutamine:D-fructose-6-phosphate aminotransferase.L-Glutamine:D-fructose-6-phosphate aminotransferase is the first enzymeof the supply pathway of UDP-N-acetylglucosamine, which is a precursorof heparosan, and catalyzes the reaction of convertingfructose-6-phosphate to glucosamine-6-phosphate. However, there has notbeen any report indicating the relationship between enhancement of theactivity of GlmS and the heparosan-producing ability, and there has notbeen any report indicating the relationship between GlmZ and heparosanproduction.

HemY, HemX, and HemD are enzymes of the biosynthetic pathways of hemeand choline. HemY is a protoporphyrinogen oxidase that oxidizesprotoporphyrinogen IX to generate protoporphyrin IX in the hemebiosynthesis pathway (Dailey T. A. et al. (1994) “Expression of a clonedprotoporphyrinogen oxidase”, The Journal of Biological Chemistry,269:813-815). Although HemX is presumed to be an uroporphyrinogen IIImethylase that methylates uroporphyrinogen III to generate precholine IIin the choline biosynthetic pathway, actual function thereof is unknown(Sasarman A. et al. (1988) “Nucleotide sequence of the hemX gene, thethird member of the Uro operon of Escherichia coli K12”, Nucleic AcidsRes., 16(24):11835). HemD is an uroporphyrinogen III synthase thatgenerates uroporphyrinogen III, which is the last common metabolicintermediate in the biosynthetic pathways of heme and choline (Jordan P.M. and Woodcock S. C. (1991) “Mutagenesis of arginine residues in thecatalytic cleft of Escherichia coli porphobilinogen deaminase thataffects dipyrromethane cofactor assembly and tetrapyrrole chaininitiation and elongation”, Biochem. J., 280(Pt 2):445-9). There has notbeen any report indicating the relationship between these proteins andheparosan production.

RlmL (synonym is RlmKL) is a methyltransferase that methylates G2445 andG2069 of 23S rRNA (Kimura S. et al. (2012) “Base methylations in thedouble-stranded RNA by a fused methyltransferase bearing unwindingactivity”, Nucleic Acids Res., 40(9):4071-85). RlmL is a fused protein,and the N-terminus domain may be especially referred to as RlmL, and theN-terminus domain as RlmK. There has not been any report indicating therelationship between this protein and heparosan production.

ArtQ, ArtM, and ArtJ are subunits of an arginine ABC transporter (LintonK. J. and Higgins C. F. (1998) “The Escherichia coli ATP-bindingcassette (ABC) proteins”, Mol. Microbiol., 28(1):5-13). It is estimatedthat ArtJ localizes in the periplasm. Since ArtM and Art are hydrophobicproteins, it is estimated that they localize in the inner membrane, andcooperate with ArtP, which is ATPase, to function as an inner membranepenetration device for arginine. There has not been any reportindicating the relationship between these proteins and heparosanproduction.

RlmC (synonym is RumB) is a methyltransferase that methylates U747 of23S rRNA (Madsen C. T. et al. (2003) “Identifying the methyltransferasesfor m(5)U747 and m(5)U1939 in 23S rRNA using MALDI mass spectrometry”,Nucleic Acids Res., 31(16):4738-46). There has not been any reportindicating the relationship between this protein and heparosanproduction.

Although YbjO is presumed to be an inner membrane protein, its functionis unknown (Rapp M. et al. (2004) “Experimentally based topology modelsfor E. coli inner membrane proteins”, Protein Sci., 13(4):937-45). Therehas not been any report indicating the relationship between this proteinand heparosan production.

YejO is an outer membrane protein, and has a function for phase-variableprotein efflux (Henderson I. R. and Owen P. (1999) “The majorphase-variable outer membrane protein of Escherichia coli structurallyresembles the immunoglobulin A1 protease class of exported protein andis regulated by a novel mechanism involving Dam and oxyR”, J.Bacteriol., 181(7):2132-41). There has not been any report indicatingthe relationship between this protein and heparosan production.

YejM is presumed to be a hydrolase, but its actual function is unknown.Therefore, there has not been any report indicating the relationshipbetween this protein and heparosan production.

YejL is a protein of unknown function. Therefore, there has not been anyreport indicating the relationship between this protein and heparosanproduction.

RpoS is sigma S (σ^(s)), which is a sigma factor and acts as a subunitof RNA polymerase. RpoS globally controls expression of genes inresponse to stress (Maciag A. et al (2011) “In vitro transcriptionprofiling of the σS subunit of bacterial RNA polymerase: Re-definitionof the σS regulon and identification of σS-specific promoter sequenceelements”, Nucleic Acids Res., 39(13):5338-55). There has not been anyreport indicating the relationship between this protein and heparosanproduction.

YgbN is a protein presumed to be a transporter belonging to the Gntfamily involved in the gluconic acid transport, and has been suggestedto possibly be a proton-driven type metabolite uptake carrier (PeekhausN. et al. (1997) “Characterization of a novel transporter family thatincludes multiple Escherichia coli gluconate transporters and theirhomologues”, FEMS Microbiol. Lett., 147(2):233-8). There has not beenany report indicating the relationship between this protein andheparosan production.

YgbM is a protein of unknown function. Therefore, there has not been anyreport indicating the relationship between this protein and heparosanproduction.

Although YbgL is presumed to be an aldolase, actual function thereof isunknown. Therefore, there has not been any report indicating therelationship between this protein and heparosan production.

G3798 is a protein presumed to be an SOS-response transcriptionalrepressor (RecA-mediated autopeptidase). G3794 is a protein presumed tobe a superinfection exclusion protein B. G3793 is a protein presumed tobe a restriction inhibitor protein ral (antirestriction protein). Therehas not been any report indicating the relationship between theseproteins and heparosan production.

G3797, G3796, G3795, and G3792 are proteins of unknown function.Therefore, there has not been any report indicating the relationshipbetween these proteins and heparosan production.

RyjA is a low molecular weight RNA of about 140 nt (Wassarman K. M. etal. (2001) “Identification of novel small RNAs using comparativegenomics and microarrays”, Genes, Dev. 15(13):1637-51). There has notbeen any report indicating the relationship between this RNA andheparosan.

SoxRS are transcriptional control factors that participate in responseto oxidation stress. SoxR is activated by oxidation stress, and inducesexpression of SoxS, and SoxRS induce expression of the SoxRS regulongene (Gu M. and Imlay J. A. (2011) “The SoxRS response of Escherichiacoli is directly activated by redox-cycling drugs rather than bysuperoxide”, Mol. Microbiol., 79(5):1136-50; Touati D. (2000) “Sensingand protecting against superoxide stress in Escherichia coli—how manyways are there to trigger soxRS response?”, Redox Rep., 5(5):287-93).SoxRS are known to participate in generation of lipopolysaccharide (LeeJ. H. et al. (2009) “SoxRS-mediated lipopolysaccharide modificationenhances resistance against multiple drugs in Escherichia coli”, J.Bacteriol., 191(13):4441-50). However, there has not been any reportindicating the relationship between these proteins and heparosanproduction.

YjcC is a c-di-GMP-specific phosphodiesterase (Boehm A. et al. (2009)“Second messenger signaling governs Escherichia coli biofilm inductionupon ribosomal stress”, Mol. Microbiol., 72(6):1500-16). Although it isknown that overexpression of YjcC decreases biofilm formation, there hasnot been any report indicating the relationship between this protein andheparosan production.

YjcB is a protein of unknown function. Therefore, there has not been anyreport indicating the relationship between this protein and heparosanproduction.

EfeU and EfeO are components of the divalent iron ion transport carrierEfeUOB. EfeU functions as a permease, and EfeO functions as a proteinlocalizing in the periplasm (Cao J. et al. (2007) “EfeUOB (YcdNOB) is atripartite, acid-induced and CpxAR-regulated, low-pH Fe²⁺ transporterthat iscryptic in Escherichia coli K-12 but functional in E. coliO-157:H7”, Mol. Microbiol., 65:857-875). There has not been any reportindicating the relationship between these proteins and heparosanproduction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the wild-type nlpD promoter (Pn1p0). Thenucleotide sequence shown in the figure is shown as SEQ ID NO: 165.

FIG. 2 shows the structure of a variant type nlpD promoter (Pn1p8). Thenucleotide sequence shown in the figure is shown as SEQ ID NO: 168.

DETAILED DESCRIPTION OF THE INVENTION

Hereafter, the present invention will be explained in detail.

<1> Bacterium of the Present Invention

The bacterium of the present invention is an Escherichia bacteriumhaving a heparosan-producing ability that has been modified so thatexpression of one or more of the genes depicted in Tables 1 to 3 isincreased.

<1-1> Bacterium Having Heparosan-Producing Ability

the phrase “bacterium having a heparosan-producing ability” can refer toa bacterium having an ability to produce and accumulate heparosan in amedium in such a degree that heparosan can be collected, when thebacterium is cultured in the medium. The bacterium having aheparosan-producing ability may be a bacterium that is able toaccumulate heparosan in a medium in an amount larger than thatobtainable with a non-modified strain. Examples of the non-modifiedstrain include wild-type strains and parent strains of the bacterium.The bacterium having a heparosan-producing ability may be a bacteriumthat is able to accumulate heparosan in a medium in an amount of, forexample, 50 mg/L or more, 100 mg/L or more, 200 mg/L or more, or 300mg/L or more.

The Escherichia bacterium is not particularly limited, and examplesthereof include those classified into the genus Escherichia according tothe taxonomy known to those skilled in the field of microbiology.Examples of the Escherichia bacterium include, for example, thosedescribed in the work of Neidhardt et al. (Backmann B. J., 1996,Derivations and Genotypes of some mutant derivatives of Escherichia coliK-12, pp. 2460-2488, Table 1, In F. D. Neidhardt (ed.), Escherichia coliand Salmonella Cellular and Molecular Biology, Second Edition, AmericanSociety for Microbiology Press, Washington, D.C.). Examples ofEscherichia bacterium include, for example, Escherichia coli. Specificexamples of Escherichia coli include, for example, Escherichia coli K-12strains such as Escherichia coli W3110 strain (ATCC 27325) and MG1655strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506);Escherichia coli B strains such as BL21(DE3) strain; and theirderivative strains.

These strains are available from, for example, the American Type CultureCollection (Address: 12301 Parklawn Drive, Rockville, Md. 20852, P.O.Box 1549, Manassas, Va. 20108, United States of America). That is,registration numbers are given to the respective strains, and thestrains can be ordered by using these registration numbers (refer toatcc.org/). The registration numbers of the strains are listed in thecatalogue of the American Type Culture Collection. The BL21(DE3) strainis also available from, for example, Life Technologies (product numberC6000-03).

The bacterium of the present invention may be a bacterium inherentlyhaving a heparosan-producing ability, or may be a bacterium modified sothat it has a heparosan-producing ability. The bacterium having aheparosan-producing ability can be obtained by, for example, imparting aheparosan-producing ability to such a bacterium as mentioned above.

A heparosan-producing ability can be imparted by introducing a geneencoding a protein that participates in heparosan production. Examplesof such a protein that participates in heparosan production includeglycosyltransferase and heparosan efflux carrier protein. In the presentinvention, one gene or two or more different genes may be introduced. Agene may be introduced in the same manner as that of the method ofincreasing copy number of gene described later.

The term “glycosyltransferase” referred to herein means a protein havingan activity for catalyzing a reaction of adding N-acetyl-D-glucosamine(GlcNAc) and/or glucuronic acid (GlcUA) to a non-reducing end of a sugarchain to thereby extend a heparosan chain. This activity is alsoreferred to as “glycosyltransferase activity”. Examples of the geneencoding glycosyltransferase include the kfiA gene, kfiC gene, and pmHS1gene.

Examples of the kfiA gene and kfiC gene include the kfiA gene and kfiCgene of the Escherichia coli K5 strain. The KfiA protein encoded by thekfiA gene of the Escherichia coli K5 strain adds GlcNAc to anon-reducing end of a sugar chain by using UDP-GlcNAc as a substrate.The KfiC protein encoded by the kfiC gene of the Escherichia coli K5strain adds GlcUA to a non-reducing end of a sugar chain by usingUDP-GlcUA as a substrate. The kfiA and kfiC genes of the Escherichiacoli K5 strain constitute the kfiABCD operon (also referred to as region2) together with the kfiB and kfiD genes. The nucleotide sequence of aregion containing the kfiABCD operon of the Escherichia coli K5 strainis shown as SEQ ID NO: 24. In the nucleotide sequence of SEQ ID NO: 24,the kfiA, kfiB, kfiC, and kfiD genes correspond to the sequence of thepositions 445 to 1,164, the sequence of the positions 1,593 to 3,284,the sequence of the positions 4,576 to 6,138, and the sequence of thepositions 6,180 to 7,358, respectively. The amino acid sequences ofKfiA, KfiB, KfiC, and KfiD proteins of the Escherichia coli K5 strainare shown as SEQ ID NOS: 25 to 28, respectively.

Examples of the pmHS1 gene include the pmHS1 gene of the Pasteurellamultocida type D strain. The PmHS1 protein encoded by the pmHS1 gene ofthe Pasteurella multocida type D strain alternately adds GlcNAc andGlcUA to a non-reducing end of a sugar chain by using both UDP-GlcNAcand UDP-GlcUA as substrates. The nucleotide sequence of the pmHS1 geneof the Pasteurella multocida type D strain and the amino acid sequenceof the protein encoded by this gene can be obtained from publicdatabases such as the NCBI database (ncbi.nlm.nih.gov/).

The phrase “heparosan efflux carrier protein” can mean a protein havingthe activity of excreting a heparosan chain out of a cell through cellmembranes. This activity can also referred to as “heparosan effluxactivity”. Examples of a gene encoding the heparosan efflux carrierprotein include the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes.Examples of the kpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes include thekpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of the Escherichia coli K5strain and Escherichia coli B strain. The kpsC, kpsD, kpsE, and kpsSgenes of these strains constitute the kpsFEDUCS operon (also referred toas region 1) together with the kpsF and kpsU genes. The kpsM and kpsTgenes of these strains constitute the kpsMT operon (also referred to asregion 3). The nucleotide sequences of the kpsC, kpsD, kpsE, kpsM, kpsS,and kpsT genes of these strains, and the amino acid sequences of theproteins encoded by these genes can be obtained from public databasessuch as the NCBI database (ncbi.nlm.nih.gov/).

The gene to be introduced can be appropriately chosen according to typeof the bacterium to be used, and so forth. For example, the Escherichiacoli B strain has genes encoding a heparosan efflux carrier protein, butit does not have genes encoding glycosyltransferase. Therefore, aheparosan-producing ability can be imparted to the Escherichia coli Bstrain by introducing gene(s) encoding glycosyltransferase. Furthermore,for example, the Escherichia coli K-12 strain does not have either genesencoding glycosyltransferase or genes encoding a heparosan effluxcarrier protein. Therefore, a heparosan-producing ability can beimparted to the Escherichia coli K-12 strain by introducing both gene(s)encoding glycosyltransferase and genes encoding a heparosan effluxcarrier protein.

Thus, examples of the Escherichia bacterium having a heparosan-producingability include, for example, Escherichia coli K5 strain; Escherichiacoli B strain such as BL21(DE3) strain introduced with the kfiA gene andkfiC gene of the Escherichia coli K5 strain; Escherichia coli K-12strain such as W3110 strain and MG1655 strain introduced with the kfiAgene and kfiC gene of the Escherichia coli K5 strain, as well as thekpsC, kpsD, kpsE, kpsM, kpsS, and kpsT genes of the Escherichia coli K5strain or Escherichia coli B strain; and derivative strains thereof.Specific examples of Escherichia coli B strain introduced with the kfiAgene and kfiC gene of the Escherichia coli K5 strain include, forexample, the Escherichia coli BL21(DE3)/pVK9-region2 described inExamples.

The bacterium having a heparosan-producing ability may also be abacterium that has been modified so that expression is enhanced of agene encoding a protein involved in the heparosan production and that isinherently present in the bacterium. That is, for example, theEscherichia coli K5 strain may be modified so that expression of one ormore genes encoding a protein that participates in the heparosanproduction is enhanced. Furthermore, for example, the Escherichia coli Bstrain may be modified so that expression of one or more genes encodinga heparosan efflux carrier protein is enhanced.

The bacterium having a heparosan-producing ability may have been furthermodified in other ways so long as the heparosan-producing ability is notdegraded. For example, the bacterium having a heparosan-producingability may have been modified so that expression of one or more genes,such as kfiB, kfiD, kpsF, and kpsU, is enhanced. That is, when genesencoding glycosyltransferase are introduced, for example, region 2 maybe entirely introduced, and when genes encoding glycosyltransferase andgenes encoding a heparosan efflux carrier protein are introduced,regions 1 to 3 may be entirely introduced.

The gene used to modify a bacterium, so that, for example, impartationof a heparosan-producing ability, is not limited to the genesexemplified above or genes having a known nucleotide sequence, but maybe a variant of such genes, so long as the variant encodes a proteinthat maintains the original function. The expression “protein maintainsthe original function” means that, in the case of theglycosyltransferase, for example, the variant of the protein has theglycosyltransferase activity, or in the case of the heparosan effluxcarrier protein, the variant of the protein has the heparosan effluxactivity. For example, the gene used for modification of the bacteriumsuch as impartation of a heparosan-producing ability may be a geneencoding a protein having a known amino acid sequence includingsubstitution, deletion, insertion, or addition of one or several aminoacid residues at one or several positions. To variants of genes orproteins, the descriptions for conservative variants of the genesdepicted in Tables 1 to 3 and the proteins encoded by them can besimilarly applied.

<1-2> Increase in Expression of Genes Depicted in Tables 1 to 3

The bacterium of the present invention has been modified so thatexpression of one or more genes such as those depicted in Tables 1 to 3is increased. The bacterium of the present invention can be obtained bymodifying a bacterium having a heparosan-producing ability so thatexpression of one or more genes such as those depicted in Tables 1 to 3is increased. The bacterium of the present invention can also beobtained by modifying a bacterium so that expression of one or moregenes such as those depicted in Tables 1 to 3 is increased, and thenimparting a heparosan-producing ability to the bacterium. The bacteriumof the present invention may be a bacterium that has acquired aheparosan-producing ability as a result of the modification forincreasing expression of one or more genes such as those depicted inTables 1 to 3. In the present invention, modifications for constructingthe bacterium of the present invention can be performed in an arbitraryorder.

The “genes depicted in Tables 1 to 3” are, specifically, rbsR, rbsK,rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB,rfaH, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA,g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK,rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU,paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, lepB, rnc, era,dapA, gcvR, bcp, hyfA, rpoE, nadB, yfiC, srmB, g1414, g1413, nuoE, nuoF,nuoG, glmZ, hemY, hemX, hemD, rlmL, artQ, artM, artJ, rlmC, ybjO, yejO,yejM, yejL, rpoS, ygbN, ygbM, ygbL, g3798, g3797, g3796, g3795, g3794,g3793, g3792, ryjA, soxR, soxS, yjcC, yjcB, efeU, and efeO. The genesdepicted in Tables 1 to 3 are also referred to as “the genes of Tables 1to 3”.

The rbsR, rbsK, and rbsB genes are genes encoding factors thatparticipate in uptake of D-ribose. The rbsR gene encodes the repressorof the rbs operon. The rbsK gene encodes a ribokinase. The rbsB geneencodes one of the subunits of the ribose ABC transporter. The rbsR,rbsK, and rbsB genes of the Escherichia coli K-12 MG1655 straincorrespond to the sequence of the positions 3,936,250 to 3,937,242, thesequence of the positions 3,935,317 to 3,936,246, and the sequence ofthe positions 3,934,301 to 3,935,191 of the genome sequence registeredat the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990), respectively. The RbsR, RbsK, and RbsB proteins of theMG1655 strain are registered as GenBank accession NP 418209 (versionNP_418209.1 GI: 16131621), GenBank accession NP_418208 (versionNP_418208.1 GI: 16131620), and GenBank accession NP_418207 (versionNP_418207.1 GI: 16131619), respectively.

The hsrA gene encodes an inner membrane protein presumed to be a memberof the major facilitator superfamily (MFS). The hsrA gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 3,937,208 to 3,938,635 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The HsrA protein of theMG1655 strain is registered as GenBank accession NP_418210 (versionNP_418210.1 GI: 16131622).

The nucleotide sequence of a region containing the rbsB, rbsK, rbsR, andhsrA genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 29.In the nucleotide sequence shown as SEQ ID NO: 29, the rbsB, rbsK, andrbsR genes correspond to the sequence of the positions 800 to 1,690, thesequence of the positions 1,816 to 2,745, and the sequence of thepositions 2,749 to 3,741, respectively. In the nucleotide sequence shownas SEQ ID NO: 29, the hsrA gene corresponds to the complementarysequence of the sequence of the positions 3,707 to 5,134. The amino acidsequences of RbsR, RbsK, RbsB, and HsrA proteins of the Escherichia coliK5 strain are shown as SEQ ID NOS: 30 to 33, respectively.

The glgB gene encodes a glycogen branching enzyme (1,4-α-glucanbranching enzyme). The glgX gene encodes a glycogen debranching enzyme.The glgB and glgX genes of the Escherichia coli K-12 MG1655 straincorrespond to the complementary sequence of the sequence of thepositions 3,569,339 to 3,571,525, and the complementary sequence of thesequence of the positions 3,567,369 to 3,569,342 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990), respectively. The GlgB and GlgX proteins ofthe MG1655 strain are registered as GenBank accession NP_417890 (versionNP_417890.1 GI:16131306) and GenBank accession NP_417889 (versionNP_417889.1 GI: 16131305), respectively.

The nucleotide sequence of a region containing the glgB and glgX genesof the Escherichia coli K5 strain is shown as SEQ ID NO: 34. In thenucleotide sequence shown as SEQ ID NO: 34, the glgB and glgX genescorrespond to the sequence of the positions 989 to 3,175, and thesequence of the positions 3,172 to 5,145, respectively. The amino acidsequences of the GlgB and GlgX proteins of the Escherichia coli K5strain are shown as SEQ ID NOS: 35 and 36, respectively.

The micF gene encodes an antisense RNA that participates in theexpression inhibition of OmpF. The micF gene of the Escherichia coliK-12 MG1655 strain corresponds to the sequence of the positions2,311,106 to 2,311,198 in the genome sequence registered at the NCBIdatabase as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990).

The rcsD and rcsB genes encode a transcription factor. The rcsD and rcsBgenes of the Escherichia coli K-12 MG1655 strain correspond to thesequences of the positions 2,311,510 to 2,314,182, and the positions2,314,199 to 2,314,849 in the genome sequence registered at the NCBIdatabase as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990), respectively. The RcsD and RcsB proteins of the MG1655 strainare registered as GenBank accession NP_416720 (version NP_416720.1GI:16130153) and GenBank accession NP_416721 (version NP_416721.1 GI:16130154), respectively.

The nucleotide sequence of a region containing the rcsB, rcsD, and micFgenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 43. Inthe nucleotide sequence shown as SEQ ID NO: 43, the rcsB, rcsD, and micFgenes correspond to the sequence of the positions 3,312 to 3,962, thesequence of the positions 623 to 3,295, and the sequence of thepositions 219 to 311, respectively. The amino acid sequences of RcsB andRcsD proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS:44 and 45, respectively.

The ybiX, ybiI, ybiJ, ybiC, and ybiB genes are genes of unknownfunctions. The ybiX, ybiI, ybiJ, ybiC, and ybiB genes of the Escherichiacoli K-12 MG1655 strain correspond to the complementary sequence of thesequence of the positions 837,753 to 838,430, the complementary sequenceof the sequence of the positions 837,413 to 837,679, the complementarysequence of the sequence of the positions 836,888 to 837,148, thesequence of the positions 835,574 to 836,659, and the sequence of thepositions 834,471 to 835,433 in the genome sequence registered at theNCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990), respectively. The YbiX, Ybil, YbiJ, YbiC, and YbiB proteinsof the MG1655 strain are registered as GenBank accession NP_415325(version NP_415325.4 GI: 90111170), GenBank accession NP_415324 (versionNP_415324.1 GI: 16128771), GenBank accession NP_415323 (versionNP_415323.1 GI: 16128770), GenBank accession NP_415322 (versionNP_415322.1 GI: 16128769), and GenBank accession NP_415321 (versionNP_415321.1 GI: 16128768), respectively.

The nucleotide sequence of a region containing the ybiX, ybiI, ybiJ,ybiC, and ybiB genes of the Escherichia coli K5 strain is shown as SEQID NO: 37. In the nucleotide sequence shown as SEQ ID NO: 37, the ybiX,ybiI, and ybiJ genes correspond to the sequence of the positions 718 to1,395, the sequence of the positions 1,469 to 1,735, and the sequence ofthe positions 2,000 to 2,260, respectively. In the nucleotide sequenceshown as SEQ ID NO: 37, the ybiC and ybiB genes correspond to thecomplementary sequence of the sequence of the positions 2,488 to 3,574,and the complementary sequence of the sequence of the positions 3,715 to4,677. The amino acid sequences of the YbiX, Ybil, YbiJ, YbiC, and YbiBproteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 38to 42, respectively.

The rfaH and nusG genes encode a transcription factor. The rfaH and nusGgenes of the Escherichia coli K-12 MG1655 strain correspond to thecomplementary sequence of the sequence of the positions 4,022,356 to4,022,844, and the sequence of the positions 4,175,766 to 4,176,311 inthe genome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The RfaH andNusG proteins of the MG1655 strain are registered as GenBank accessionNP_418284 (version NP_418284.1 GI:16131688) and GenBank accessionNP_418409 (version NP_418409.1 GI: 16131812), respectively.

The nucleotide sequence of the rfaH gene of the Escherichia coliBL21(DE3) strain, and the amino acid sequence of the RfaH proteinencoded by this gene are shown as SEQ ID NOS: 46 and 47, respectively.The nucleotide sequence of the nusG gene of the Escherichia coliBL21(DE3) strain, and the amino acid sequence of the NusG proteinencoded by this gene are shown as SEQ ID NOS: 48 and 49, respectively.

The pcoR, pcoS, and pcoE genes are genes encoding a factor that relatesto copper resistance. The pcoR gene encodes a protein homologous to theactivator of the pco operon. The pcoS gene encodes a protein homologousto the sensor protein of a two-component control system. The pcoE geneencodes a copper binding protein. These genes are not annotated in thegenome of the Escherichia coli K-12 MG1655 strain.

The nucleotide sequence of a region containing the pcoR, pcoS, and pcoEgenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 50. Inthe nucleotide sequence shown as SEQ ID NO: 50, the pcoR, pcoS, and pcoEgenes correspond to the sequence of the positions 128 to 808, thesequence of the positions 805 to 2,205, and the sequence of thepositions 2,423 to 2,857, respectively. The amino acid sequences of thePcoR, PcoS, and PcoE proteins of the Escherichia coli K5 strain areshown as SEQ ID NOS: 51 to 53, respectively.

The yhcN gene encodes a factor that participates in stress response. TheyhcN gene of the Escherichia coli K-12 MG1655 strain corresponds to thesequence of the positions 3,383,560 to 3,383,823 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The YhcN protein of the MG1655 strain isregistered as GenBank accession NP_417705 (version NP_417705.2 GI:90111561).

The yhcO gene encodes a protein homologous to an inhibitor for RNase.The yhcO gene of the Escherichia coli K-12 MG1655 strain corresponds tothe complementary sequence of the sequence of the positions 3,383,879 to3,384,151 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YhcOprotein of the MG1655 strain is registered as GenBank accessionNP_417706 (version NP_417706.1 GI: 16131129).

The aaeB and aaeA genes encode a subunit of an efflux carrier of4-hydroxybenzoic acid. The aaeX gene encodes a protein presumed to be anefflux carrier. The aaeB, aaeA, and aaeX genes of the Escherichia coliK-12 MG1655 strain correspond to the complementary sequence of thesequence of the positions 3,384,243 to 3,386,210, the complementarysequence of the sequence of the positions 3,386,216 to 3,387,148, andthe complementary sequence of the sequence of the positions 3,387,156 to3,387,359 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990),respectively. The AaeB, AaeA, and AaeX proteins of the MG1655 strain areregistered as GenBank accession NP_417707 (version NP_417707.1 GI:16131130), GenBank accession NP_417708 (version NP_417708.1 GI:16131131), and GenBank accession NP_417709 (version NP_417709.2 GI:90111562), respectively.

The nucleotide sequence of a region containing the yhcN, yhcO, aaeB,aaeA, and aaeX genes of the Escherichia coli K5 strain is shown as SEQID NO: 54. In the nucleotide sequence shown as SEQ ID NO: 54, the yhcN,yhcO, aaeB, aaeA, and aaeX genes correspond to the sequence of thepositions 63 to 326, the complementary sequence of the sequence of thepositions 382 to 654, the complementary sequence of the sequence of thepositions 746 to 2,713, the complementary sequence of the sequence ofthe positions 2,719 to 3,651, and the complementary sequence of thesequence of the positions 3,659 to 3,931, respectively. The amino acidsequences of the YhcN, YhcO, AaeB, AaeA, and AaeX proteins of theEscherichia coli K5 strain are shown as SEQ ID NOS: 55 to 59,respectively.

The g1455 and g1453 genes are genes of unknown functions. These genesare not annotated in the genome of the Escherichia coli K-12 MG1655strain.

The alpA gene encodes an expression control factor of the intA gene. ThealpA gene of the Escherichia coli K-12 MG1655 strain corresponds to thesequence of the positions 2,756,666 to 2,756,878 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The AlpA protein of the MG1655 strain isregistered as GenBank accession NP_417113 (version NP_417113.1 GI:16130542).

The nucleotide sequence of a region containing the g1455, alpA, andg1453 genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 60.In the nucleotide sequence shown as SEQ ID NO: 60, the g1455, alpA, andg1453 genes correspond to the complementary sequence of the sequence ofthe positions 568 to 1,140, the complementary sequence of the sequenceof the positions 1,226 to 1,486, and the sequence of the positions 2,389to 2,529, respectively. The amino acid sequences of G1455, AlpA, andG1453 proteins of the Escherichia coli K5 strain are shown as SEQ IDNOS: 61 to 63, respectively.

The yrbA gene (synonym is ibaG) encodes a protein presumed to be aDNA-binding type transcription factor. The yrbA gene of the Escherichiacoli K-12 MG1655 strain corresponds to the complementary sequence of thesequence of the positions 3,334,571 to 3,334,825 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The YrbA protein of the MG1655 strain isregistered as GenBank accession NP_417657 (version NP_417657.2 GI:90111555).

The mlaB, mlaC, mlaD, mlaE, and mlaF genes encode a constituent factorof a phospholipid ABC transporter. The mlaB, mlaC, mlaD, mlaE, and mlaFgenes of the Escherichia coli K-12 MG1655 strain correspond to thecomplementary sequence of the sequence of the positions 3,334,985 to3,335,278, the complementary sequence of the sequence of the positions3,335,278 to 3,335,913, the complementary sequence of the sequence ofthe positions 3,335,932 to 3,336,483, the complementary sequence of thesequence of the positions 3,336,488 to 3,337,270, and the complementarysequence of the sequence of the positions 3,337,278 to 3,338,087 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The MlaB,MlaC, MlaD, MlaE, and MlaF proteins of the MG1655 strain are registeredas GenBank accession NP_417658 (version NP_417658.4 GI: 90111556),GenBank accession NP_417659 (version NP_417659.1 GI: 16131082), GenBankaccession NP_417660 (version NP_417660.1 GI: 16131083), GenBankaccession NP_417661 (version NP_417661.1 GI: 16131084), and GenBankaccession NP_417662 (version NP_417662.1 GI: 16131085), respectively.

The yrbG gene encodes a protein presumed to be an Na⁺/Ca²⁺ antiporter.The yrbG gene of the Escherichia coli K-12 MG1655 strain corresponds tothe sequence of the positions 3,338,297 to 3,339,274 in the genomesequence registered at the NCBI database as GenBank accession NC_000913(VERSION NC_000913.2 GI: 49175990). The YrbG protein of the MG1655strain is registered as GenBank accession NP_417663 (version NP_417663.1GI: 16131086).

The nucleotide sequence of a region containing the yrbA, mlaB, mlaC,mlaD, mlaE, mlaF, and yrbG genes of the Escherichia coli K5 strain isshown as SEQ ID NO: 64. In the nucleotide sequence shown as SEQ ID NO:64, the yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, and yrbG genes correspond tothe complementary sequence of the sequence of the positions 977 to1,246, the complementary sequence of the sequence of the positions 1,391to 1,780, the complementary sequence of the sequence of the positions1,684 to 2,319, the complementary sequence of the sequence of thepositions 2,338 to 2,889, the complementary sequence of the sequence ofthe positions 2,894 to 3,676, the complementary sequence of the sequenceof the positions 3,684 to 4,493, and the sequence of the positions 4,703to 5,680, respectively. The amino acid sequences of YrbA, MlaB, MlaC,MlaD, MlaE, MlaF, and YrbG proteins of the Escherichia coli K5 strainare shown as SEQ ID NOS: 65 to 71, respectively.

The norW gene encodes an NO reductase. The norW gene of the Escherichiacoli K-12 MG1655 strain corresponds to the sequence of the positions2,831,934 to 2,833,067 in the genome sequence registered at the NCBIdatabase as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990). The NorW protein of the MG1655 strain is registered asGenBank accession NP_417191 (version NP_417191.1 GI: 16130618).

The nucleotide sequence of a region containing the norW gene of theEscherichia coli K5 strain is shown as SEQ ID NO: 72. In the nucleotidesequence shown as SEQ ID NO: 72, the norW gene corresponds to thesequence of the positions 1,201 to 2,334. The amino acid sequence of theNorW protein of the Escherichia coli K5 strain is shown as SEQ ID NO:73.

The ybjI gene encodes a flavin mononucleotide (FMN) phosphorylatingenzyme. The ybjI gene of the Escherichia coli K-12 MG1655 straincorresponds to the complementary sequence of the sequence of thepositions 884,539 to 885,354 in the genome sequence registered at theNCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990). The YbjI protein of the MG1655 strain is registered asGenBank accession NP_415365 (version NP_415365.4 GI: 90111176).

The ybjJ and ybjK genes are genes of unknown function. The ybjJ and ybjKgenes of the Escherichia coli K-12 MG1655 strain correspond to thecomplementary sequence of the sequence of the positions 885,354 to886,562, and the sequence of the positions 886,646 to 887,182 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The YbjJ andYbjK proteins of the MG1655 strain are registered as GenBank accessionNP_415366 (version NP_415366.1 GI:16128813) and GenBank accessionNP_415367 (version NP_415367.1 GI:16128814), respectively.

The rybB gene encodes a low molecular weight RNA that participates inexpression inhibition of OmpC and OmpW. The rybB gene of the Escherichiacoli K-12 MG1655 strain corresponds to the complementary sequence of thesequence of the positions 887,199 to 887,277 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990).

The nucleotide sequence of a region containing the ybjI, ybjJ, ybjK, andrybB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 74.In the nucleotide sequence shown as SEQ ID NO: 74, the ybjI, ybjJ, ybjK,and rybB genes correspond to the complementary sequence of the sequenceof the positions 117 to 932, the complementary sequence of the sequenceof the positions 932 to 2,140, the sequence of the positions 2,224 to2,760, and the complementary sequence of the sequence of the positions2,777 to 2,855, respectively. The amino acid sequences of the YbjI,YbjJ, and YbjK proteins of the Escherichia coli K5 strain are shown asSEQ ID NOS: 75 to 77, respectively.

The yjjY gene is a gene of unknown function. The yjjY gene of theEscherichia coli K-12 MG1655 strain corresponds to the sequence of thepositions 4,638,425 to 4,638,565 in the genome sequence registered atthe NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990). The YjjY protein of the MG1655 strain is registered asGenBank accession NP_418819 (version NP_418819.1 GI: 16132219).

The yjtD gene encodes a protein presumed to be one of RNAmethyltransferases. The yjtD gene of the Escherichia coli K-12 MG1655strain corresponds to the sequence of the positions 4,638,965 to4,639,651 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YjtDprotein of the MG1655 strain is registered as GenBank accessionNP_418820 (version NP_418820.1 GI: 16132220).

The thrL, thrA, and thrB genes encode an enzyme of the threoninebiosynthesis pathway. The thrB gene encodes a homoserine kinase. ThethrA gene encodes an enzyme having two functions of aspartate kinase Iand homoserine dehydrogenase I. The thrL gene encodes a leader peptideof the thrLABC operon. The thrL, thrA, and thrB genes of the Escherichiacoli K-12 MG1655 strain correspond to the sequence of the positions 190to 255, the sequence of the positions 337 to 2,799, and the sequence ofthe positions 2,801 to 3,733 in the genome sequence registered at theNCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990), respectively. The ThrL, ThrA, and ThrB proteins of the MG1655strain are registered as GenBank accession NP_414542 (versionNP_414542.1 GI: 16127995), GenBank accession NP_414543 (versionNP_414543.1 GI: 16127996), and GenBank accession NP_414544 (versionNP_414544.1 GI: 16127997), respectively.

The nucleotide sequence of a region containing the yjjY, yjtD, thrL,thrA, and thrB genes of the Escherichia coli K5 strain is shown as SEQID NO: 78. In the nucleotide sequence shown as SEQ ID NO: 78, the yjjY,yjtD, thrL, thrA, and thrB genes correspond to the sequence of thepositions 124 to 264, the sequence of the positions 664 to 1,350, thesequence of the positions 1,564 to 1,629, the sequence of the positions1,711 to 4,173, and the sequence of the positions 4,175 to 5,107,respectively. The amino acid sequences of the YjjY, YjtD, ThrL, ThrA,and ThrB proteins of the Escherichia coli K5 strain are shown as SEQ IDNOS: 79 to 83, respectively.

The fruA gene encodes a fructose PTS permease. The fruA gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 2,257,741 to 2,259,432 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The FruA protein of theMG1655 strain is registered as GenBank accession NP_416672 (versionNP_416672.1 GI: 16130105).

The psuK gene encodes a pseudouridine kinase. The psuK gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 2,256,377 to 2,257,318 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The PsuK protein of theMG1655 strain is registered as GenBank accession NP_416671 (versionNP_416671.1 GI: 16130104).

The nucleotide sequence of a region containing the fruA and psuK genesof the Escherichia coli K5 strain is shown as SEQ ID NO: 84. In thenucleotide sequence shown as SEQ ID NO: 84, the fruA and psuK genescorrespond to the sequence of the positions 897 to 2588, and thesequence of the positions 3,165 to 3,953, respectively. The amino acidsequences of the FruA and PsuK proteins of the Escherichia coli K5strain are shown as SEQ ID NOS: 85 and 86, respectively.

The ytfT and yjfF genes are genes encoding a protein presumed to be amembrane constituent component of the galactose ABC transport carrier.The ytfT and yjfF genes of the Escherichia coli K-12 MG1655 straincorrespond to the sequence of the positions 4,450,594 to 4,451,619 andthe sequence of the positions 4,451,606 to 4,452,601 in the genomesequence registered at the NCBI database as GenBank accession NC_000913(VERSION NC_000913.2 GI: 49175990), respectively. The YtfT and YjfFproteins of the MG1655 strain are registered as GenBank accessionNP_418651 (version NP_418651.3 GI:145698343) and GenBank accessionNP_418652 (version NP_418652.2 GI: 90111710), respectively.

The fbp gene encodes a fructose-1,6-diphosphate phosphatase(fructose-1,6-bisphosphatase). The fbp gene of the Escherichia coli K-12MG1655 strain corresponds to the complementary sequence of the sequenceof the positions 4,452,634 to 4,453,632 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The Fbp protein of the MG1655 strain isregistered as GenBank accession NP_418653 (version NP_418653.1 GI:16132054).

The nucleotide sequence of a region containing the ytfT, yjfF, and fbpgenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 87. Inthe nucleotide sequence shown as SEQ ID NO: 87, the ytfT, yjfF, and fbpgenes correspond to the sequence of the positions 252 to 1,277, thesequence of the positions 1,264 to 2,259, and the complementary sequenceof the sequence of the positions 2,292 to 3,290, respectively. The aminoacid sequences of the YtfT, YjfF, and Fbp proteins of the Escherichiacoli K5 strain are shown as SEQ ID NOS: 88 to 90, respectively.

The yagU gene encodes a protein presumed to be an inner membraneprotein. The yagU gene of the Escherichia coli K-12 MG1655 straincorresponds to the sequence of the positions 302,215 to 302,829 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The YagU protein of theMG1655 strain is registered as GenBank accession NP_414821 (versionNP_414821.1 GI: 16128272).

The paoA gene (also called yagT) and paoB gene (also called yagS) aregenes encoding a constituent factor of an aldehyde oxidoreductase. ThepaoA and paoB genes of the Escherichia coli K-12 MG1655 straincorrespond to the complementary sequence of the sequence of thepositions 301,108 to 301,797, and the complementary sequence of thesequence of the positions 300,155 to 301,111 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990), respectively. The PaoA and PaoB proteins ofthe MG1655 strain are registered as GenBank accession NP_414820 (versionNP_414820.1 GI:16128271) and GenBank accession NP_414819 (versionNP_414819.1 GI: 16128270), respectively.

The nucleotide sequence of a region containing the yagU, paoA, and paoBgenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 91. Inthe nucleotide sequence shown as SEQ ID NO: 91, the yagU, paoA, and paoBgenes correspond to the complementary sequence of the sequence of thepositions 117 to 731, the sequence of the positions 1,149 to 1,838, andthe sequence of the positions 1,835 to 2,791, respectively. The aminoacid sequences of the YagU, PaoA, and PaoB proteins of the Escherichiacoli K5 strain are shown as SEQ ID NOS: 92 to 94, respectively.

The gsiC and gsiD genes are genes encoding a constituent factor of aglutathione ABC transport carrier. The gsiC and gsiD genes of theEscherichia coli K-12 MG1655 strain correspond to the sequence of thepositions 870,190 to 871,110, and the sequence of the positions 871,113to 872,024 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990),respectively. The GsiC and GsiD proteins of the MG1655 strain areregistered as GenBank accession NP_415352 (version NP_415352.1GI:16128799) and GenBank accession NP_415353 (version NP_415353.1 GI:16128800), respectively.

The yliE gene encodes a protein presumed to be a c-di-GMP-specificphosphodiesterase. The yliE gene of the Escherichia coli K-12 MG1655strain corresponds to the sequence of the positions 872,202 to 874,550in the genome sequence registered at the NCBI database as GenBankaccession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YliE proteinof the MG1655 strain is registered as GenBank accession NP_415354(version NP_415354.1 GI: 16128801).

The nucleotide sequence of a region containing the gsiC, gsiD, and yliEgenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 95. Inthe nucleotide sequence shown as SEQ ID NO: 95, the gsiC, gsiD, and yliEgenes correspond to the sequence of the positions 264 to 1,184, thesequence of the positions 1,187 to 2,098, and the sequence of thepositions 2,276 to 4,624, respectively. The amino acid sequences of theGsiC, GsiD, and YliE proteins of the Escherichia coli K5 strain areshown as SEQ ID NOS: 96 to 98, respectively.

The irp2 and irp1 genes encode a non-ribosormal peptide synthetase. Theirp2 and irp1 genes are not annotated in the genome of the Escherichiacoli K-12 MG1655 strain. In the present invention, the irp2 and irp1gene may be generically called “irp gene”.

The nucleotide sequence of a region containing a part of the irp gene ofthe Escherichia coli K5 strain is shown as SEQ ID NO: 99. This regioncontains the second half moiety of the irp2 gene (moiety of thepositions 2,781 to 6,108 in the full length of 6108 bp, equivalent toabout 54% of the full length), and the first half moiety of the irp1gene (moiety of the positions 1 to 2,530 in the full length of 9492 bp,equivalent to about 27% of the full length). The nucleotide sequence ofthe irp2 gene of the Escherichia coli K5 strain and the amino acidsequence of the Irp2 protein encoded by that gene are shown as SEQ IDNOS: 100 and 101, respectively. The nucleotide sequence of the irp1 geneof the Escherichia coli K5 strain and the amino acid sequence of theIrp1 protein encoded by that gene are shown as SEQ ID NOS: 102 and 103,respectively.

The bhsA gene (synonym is ycfR) encodes a protein presumed to be anouter membrane protein. The bhsA gene of the Escherichia coli K-12MG1655 strain corresponds to the sequence of the positions 1,168,296 to1,168,553 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The BhsAprotein of the MG1655 strain is registered as GenBank accessionNP_415630 (version NP_415630.1 GI: 16129075).

The ycJS gene encodes one of L,D-transpeptidases. The ycJS gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 1,168,635 to 1,169,597 in thegenome sequence registered at the NCBI database as GenBankaccessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The YcfS proteinof the MG1655 strain is registered as GenBank accession NP_415631(version NP_415631.1 GI: 16129076).

The nucleotide sequence of a region containing the bhsA and ycfS genesof the Escherichia coli K5 strain is shown as SEQ ID NO: 104. In thenucleotide sequence shown as SEQ ID NO: 104, the bhsA and ycJS genescorrespond to the sequence of the positions 440 to 697, and thecomplementary sequence of the sequence of the positions 779 to 1,741,respectively. The amino acid sequences of the BhsA and YcfS proteins ofthe Escherichia coli K5 strain are shown as SEQ ID NOS: 105 and 106,respectively.

The lepB gene encodes a signal peptidase. The lepB gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 2,702,357 to 2,703,331 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The LepB protein of theMG1655 strain is registered as GenBank accession NP_417063 (versionNP_417063.1 GI: 16130493).

The rnc gene encodes an RNaseIII. The rnc gene of the Escherichia coliK-12 MG1655 strain corresponds to the complementary sequence of thesequence of the positions 2,701,405 to 2,702,085 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The Rnc protein of the MG1655 strain isregistered as GenBank accession NP_417062 (version NP_417062.1 GI:16130492).

The era gene encodes a factor indispensable to survival. The era gene ofthe Escherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 2,700,503 to 2,701,408 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The Era protein of theMG1655 strain is registered as GenBank accession NP_417061 (versionNP_417061.1 GI: 16130491).

The nucleotide sequence of a region containing the lepB, rnc, and eragenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 107. Inthe nucleotide sequence shown as SEQ ID NO: 107, the lepB, rnc, and eragenes correspond to the sequence of the positions 1,344 to 2,318, thesequence of the positions 2,590 to 3,270, and the sequence of thepositions 3,267 to 4,172, respectively. The amino acid sequences of theLepB, Rnc, and Era proteins of the Escherichia coli K5 strain are shownas SEQ ID NOS: 108 to 110, respectively.

The dapA gene encodes a 4-hydroxy-tetrahydrodipicolinate synthase. ThedapA gene of the Escherichia coli K-12 MG1655 strain corresponds to thecomplementary sequence of the sequence of the positions 2,596,904 to2,597,782 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The DapAprotein of the MG1655 strain is registered as GenBank accessionNP_416973 (version NP_416973.1 GI: 16130403).

The gcvR gene encodes a protein presumed to be a transcription controlfactor. The gcvR gene of the Escherichia coli K-12 MG1655 straincorresponds to the sequence of the positions 2,597,928 to 2,598,500 inthe genome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The GcvR protein of theMG1655 strain is registered as GenBank accession NP_416974 (versionNP_416974.4 GI: 90111443).

The bcp gene encodes a thiol peroxidase. The bcp gene of the Escherichiacoli K-12 MG1655 strain corresponds to the sequence of the positions2,598,500 to 2,598,970 in the genome sequence registered at the NCBIdatabase as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990). The Bcp protein of the MG1655 strain is registered as GenBankaccessionNP_416975 (version NP_416975.1 GI: 16130405).

The hyfA gene encodes a protein presumed to participate in the electrontransportation. The hyfA gene of the Escherichia coli K-12 MG1655 straincorresponds to the sequence of the positions 2,599,223 to 2,599,840 inthe genome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The HyfA protein of theMG1655 strain is registered as GenBank accession NP_416976 (versionNP_416976.4 GI: 90111444).

The nucleotide sequence of a region containing the dapA, gcvR, bcp, andhyfA genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 111.In the nucleotide sequence shown as SEQ ID NO: 111, the dapA, gcvR, bcp,and hyfA genes correspond to the complementary sequence of the sequenceof the positions 858 to 1,736, the sequence of the positions 1,882 to2,454, the sequence of the positions 2,454 to 2,924, and the sequence ofthe positions 3,177 to 3,794, respectively. The amino acid sequences ofthe DapA, GcvR, Bcp, and HyfA proteins of the Escherichia coli K5 strainare shown as SEQ ID NOS: 112 to 115, respectively.

The rpoE gene encodes SigmaE (σ^(E)). The rpoE gene of the Escherichiacoli K-12 MG1655 strain corresponds to the complementary sequence of thesequence of the positions 2,707,459 to 2,708,034 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The RpoE protein of the MG1655 strain isregistered as GenBank accession NP_417068 (version NP_417068.1 GI:16130498).

The nadB gene encodes an L-aspartic acid oxidase. The nadB gene of theEscherichia coli K-12 MG1655 strain corresponds to the sequence of thepositions 2,708,442 to 2,710,064 in the genome sequence registered atthe NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990). The NadB protein of the MG1655 strain is registered asGenBank accession NP_417069 (version NP_417069.1 GI: 16130499).

The yfiC gene encodes a methyltransferase that methylates N at theposition 6 of A37 (adenine at the position 37) of valine tRNA. The yfiCgene of the Escherichia coli K-12 MG1655 strain corresponds to thecomplementary sequence of the sequence of the positions 2,710,049 to2,710,786 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YfiCprotein of the MG1655 strain is registered as GenBank accessionNP_417070 (version NP_417070.2 GI: 90111461).

The srmB gene encodes a DEAD-box type RNA helicase. The srmB gene of theEscherichia coli K-12 MG1655 strain corresponds to the sequence of thepositions 2,710,918 to 2,712,252 in the genome sequence registered atthe NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990). The SrmB protein of the MG1655 strain is registered asGenBank accession NP_417071 (version NP_417071.1 GI: 16130501).

The nucleotide sequence of a region containing the rpoE, nadB, yfiC, andsrmB genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 116.In the nucleotide sequence shown as SEQ ID NO: 116, the rpoE, nadB,yfiC, and srmB genes correspond to the complementary sequence of thesequence of the positions 355 to 930, the sequence of the positions1,338 to 2,960, the complementary sequence of the sequence of thepositions 2,945 to 3,682, and the sequence of the positions 3,814 to5,148, respectively. Among these, the nucleotide sequence of the rpoEgene of the Escherichia coli K5 strain is especially shown as SEQ ID NO:174. The amino acid sequences of the RpoE, NadB, YfiC, and SrmB proteinsof the Escherichia coli K5 strain are shown as SEQ ID NOS: 117 to 120,respectively.

The g1414 and g1413 genes are genes of unknown function. These genes arenot annotated in the genome of the Escherichia coli K-12 MG1655 strain.

The nucleotide sequence of a region containing the g1414 and g1413 genesof the Escherichia coli K5 strain is shown as SEQ ID NO: 121. In thenucleotide sequence shown as SEQ ID NO: 121, the g1414 and g1413 genescorrespond to the sequence of the positions 28 to 699, and the sequenceof the positions 831 to 1,157, respectively. The amino acid sequences ofthe G1414 and G1413 proteins of the Escherichia coli K5 strain are shownas SEQ ID NOS: 122 and 123, respectively.

The nuoE, nuoF, and nuoG genes encode a soluble fragment of NADHdehydrogenase I. The nuoE, nuoF, and nuoG genes of the Escherichia coliK-12 MG1655 strain correspond to the complementary sequence of thesequence of the positions 2,399,574 to 2,400,074, the complementarysequence of the sequence of the positions 2,398,240 to 2,399,577, andthe complementary sequence of the sequence of the positions 2,395,461 to2,398,187 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990),respectively. The NuoE, NuoF, and NuoG proteins of the MG1655 strain areregistered as GenBank accession NP_416788 (version NP_416788.1 GI:16130220), GenBank accession NP_416787 (version NP_416787.1 GI:16130219), and GenBank accession NP_416786 (version NP_416786.4 GI:145698290), respectively.

The nucleotide sequence of a region containing the nuoE, nuoF, and nuoGgenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 124. Inthe nucleotide sequence shown as SEQ ID NO: 124, the nuoE, nuoF, andnuoG genes correspond to the complementary sequence of the sequence ofthe positions 796 to 1,296, the complementary sequence of the sequenceof the positions 1,293 to 2,630, and the complementary sequence of thesequence of the positions 2,683 to 5,409, respectively. The amino acidsequences of the NuoE, NuoF, and NuoG proteins of the Escherichia coliK5 strain are shown as SEQ ID NOS: 125 to 127, respectively.

The glmZ gene encodes a low molecular weight RNA. The glmZ gene of theEscherichia coli K-12 MG1655 strain corresponds to the sequence of thepositions 3,984,455 to 3,984,626 in the genome sequence registered atthe NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990).

The hemY, hemX, and hemD genes encode enzymes of the biosynthesispathways of heme and choline. The hemY gene encodes a protoporphyrinogenoxidase. The hemX gene encodes a protein presumed to be auroporphyrinogen III methylase. The hemD gene encodes a uroporphyrinogenIII synthase. The hemY, hemX, and hemD genes of the K-12 MG1655 straincorrespond to the complementary sequence of the sequence of thepositions 3,984,709 to 3,985,905, the complementary sequence of thesequence of the positions 3,985,908 to 3,987,089, and the complementarysequence of the sequence of the positions 3,987,111 to 3,987,851 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The HemY,HemX, and HemD proteins of the MG1655 strain are registered as GenBankaccession NP_418246 (version NP_418246.1 GI: 16131654), GenBankaccession NP_418247 (version NP_418247.1 GI: 16131655), and GenBankaccession NP_418248 (version NP_418248.1 GI: 16131656), respectively.

The nucleotide sequence of a region containing the glmZ, hemY, hemX, andhemD genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 128.In the nucleotide sequence shown as SEQ ID NO: 128, the glmZ, hemY,hemX, and hemD genes correspond to the sequence of the positions 357 to563, the sequence of the positions 611 to 1,807, the sequence of thepositions 1,810 to 2,991, and the sequence of the positions 3,013 to3,753, respectively. The amino acid sequences of the HemY, HemX, andHemD proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS:129 to 131, respectively.

The rlmL gene (synonym is rlmKL) encodes a methyltransferase thatmethylates G2445 and G2069 of 23S rRNA. The rlmL gene of the Escherichiacoli K-12 MG1655 strain corresponds to the sequence of the positions1,007,067 to 1,009,175 in the genome sequence registered at the NCBIdatabase as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990). The RlmL protein of the MG1655 strain is registered asGenBank accession NP_415468 (version NP_415468.1 GI: 16128915).

The nucleotide sequence of a region containing the rlmL gene of theEscherichia coli K5 strain is shown as SEQ ID NO: 132. In the nucleotidesequence shown as SEQ ID NO: 132, the rlmL gene corresponds to thesequence of the positions 571 to 2,679. The amino acid sequence of theRlmL protein of the Escherichia coli K5 strain is shown as SEQ ID NO:133.

The artQ, artM, and artJ genes encode subunits of an arginine ABCtransporter. The artQ, artM, and artJ genes of the Escherichia coli K-12MG1655 strain correspond to the complementary sequence of the sequenceof the positions 900,757 to 901,473, the complementary sequence of thesequence of the positions 900,089 to 900,757, and the complementarysequence of the sequence of the positions 899,067 to 899,798 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990), respectively. The ArtQ,ArtM, and ArtJ proteins of the MG1655 strain are registered as GenBankaccession NP_415383 (version NP_415383.1 GI: 16128830), GenBankaccessionNP_415382 (version NP_415382.1 GI: 16128829), and GenBankaccession NP_415381 (version NP_415381.1 GI: 16128828), respectively.

The rlmC gene (synonym is rumB) encodes a methyltransferase thatmethylates U747 of 23S rRNA. The rlmC gene of the Escherichia coli K-12MG1655 strain corresponds to the sequence of the positions 897,741 to898,868 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The RlmCprotein of the MG1655 strain is registered as GenBank accessionNP_415380 (version NP_415380.1 GI: 16128827).

The ybjO gene encodes a protein presumed to be an inner membraneprotein. The ybjO gene of the Escherichia coli K-12 MG1655 straincorresponds to the sequence of the positions 897,212 to 897,700 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The YbjO protein of theMG1655 strain is registered as GenBank accession NP_415379 (versionNP_415379.1 GI: 16128826).

The nucleotide sequence of a region containing the artQ, artM, artJ,rlmC, and ybjO genes of the Escherichia coli K5 strain is shown as SEQID NO: 134. In the nucleotide sequence shown as SEQ ID NO: 134, theartQ, artM, artJ, rlmC, and ybjO genes correspond to the sequence of thepositions 386 to 1,102, the sequence of the positions 1,102 to 1,770,the sequence of the positions 2,061 to 2,792, the complementary sequenceof the sequence of the positions 2,991 to 4,118, and the complementarysequence of the sequence of the positions 4,159 to 4,647, respectively.The amino acid sequences of the ArtQ, ArtM, ArtJ, RlmC, and YbjOproteins of the Escherichia coli K5 strain are shown as SEQ ID NOS: 135to 139, respectively.

The yejO gene encodes an outer membrane protein. The yejO gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the fused sequence consisting of the sequence of thepositions 2,284,412 to 2,286,936 and the sequence of the positions2,288,136 to 2,288,202 in the genome sequence registered at the NCBIdatabase as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990). The yejO gene of the MG1655 strain is considered to be apseudogene.

The yejM gene is a gene encoding a protein presumed to be one ofhydrolases. The yejM gene of the Escherichia coli K-12 MG1655 straincorresponds to the sequence of the positions 2,282,398 to 2,284,158 inthe genome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The YejM protein of theMG1655 strain is registered as GenBank accession NP_416693 (versionNP_416693.1 GI: 16130126).

The yejL gene is a gene of unknown function. The yejL gene of theEscherichia coli K-12 MG1655 strain corresponds to the sequence of thepositions 2,282,151 to 2,282,378 in the genome sequence registered atthe NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990). The YejL protein of the MG1655 strain is registered asGenBank accession NP_416692 (version NP_416692.1 GI: 16130125).

The nucleotide sequence of a region containing the yejO, yejM, and yejLgenes of the Escherichia coli K5 strain is shown as SEQ ID NO: 140. Inthe nucleotide sequence shown as SEQ ID NO: 140, the yejO, yejM, andyejL genes correspond to the sequence of the positions 216 to 2,807, thecomplementary sequence of the sequence of the positions 3,061 to 4,821,and the complementary sequence of the sequence of the positions 4,841 to5,068, respectively. The amino acid sequences of the YejO, YejM, andYejL proteins of the Escherichia coli K5 strain are shown as SEQ ID NOS:141 to 143, respectively.

The rpoS gene encodes SigmaS (σ^(s)). The rpoS gene of the Escherichiacoli K-12 MG1655 strain corresponds to the complementary sequence of thesequence of the positions 2,864,581 to 2,865,573 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The RpoS protein of the MG1655 strain isregistered as GenBank accession NP_417221 (version NP_417221.1 GI:16130648).

The ygbN gene encodes a protein presumed to be a transporter belongingto the Gnt family. The ygbN gene of the Escherichia coli K-12 MG1655strain corresponds to the sequence of the positions 2,863,123 to2,864,487 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990). The YgbNprotein of the MG1655 strain is registered as GenBank accessionNP_417220 (version NP_417220.1 GI: 16130647).

The ygbM gene is a gene of unknown function. The ygbM gene of theEscherichia coli K-12 MG1655 strain corresponds to the sequence of thepositions 2,862,258 to 2,863,034 in the genome sequence registered atthe NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990). The YgbM protein of the MG1655 strain is registered asGenBank accession NP_417219 (version NP_417219.1 GI: 16130646).

The ygbL gene encodes a protein presumed to be one of aldolases. TheygbL gene of the Escherichia coli K-12 MG1655 strain corresponds to thesequence of the positions 2,861,615 to 2,862,253 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The YgbL protein of the MG1655 strain isregistered as GenBank accession NP_417218 (version NP_417218.1 GI:16130645).

The nucleotide sequence of a region containing the rpoS, ygbN, ygbM, andygbL genes of the Escherichia coli K5 strain is shown as SEQ ID NO: 144.In the nucleotide sequence shown as SEQ ID NO: 144, the rpoS, ygbN,ygbM, and ygbL genes correspond to the sequence of the positions 318 to1,310, the complementary sequence of the sequence of the positions 1,404to 2,768, the complementary sequence of the sequence of the positions2,857 to 3,633, and the complementary sequence of the sequence of thepositions 3,638 to 4,276, respectively. The amino acid sequences of theRpoS, YgbN, YgbM, and YgbL proteins of the Escherichia coli K5 strainare shown as SEQ ID NOS: 145 to 148, respectively.

The g3798 gene encodes a protein presumed to be an SOS-responsetranscriptional repressor (RecA-mediated autopeptidase). The g3794 geneencodes a protein presumed to be a superinfection exclusion protein B.The g3793 gene encodes a protein presumed to be a restriction inhibitorprotein ral (antirestriction protein). The g3797, g3796, g3795, andg3792 genes are genes of unknown functions. These genes are notannotated in the genome of the Escherichia coli K-12 MG1655 strain.

The nucleotide sequence of a region containing the g3798, g3797, g3796,g3795, g3794, g3793, and g3792 genes of the Escherichia coli K5 strainis shown as SEQ ID NO: 149. In the nucleotide sequence shown as SEQ IDNO: 149, the g3798, g3797, g3796, g3795, g3794, g3793, and g3792 genescorrespond to the sequence of the positions 615 to 1,268, the sequenceof the positions 1,368 to 2,219, the sequence of the positions 2,257 to2,748, the sequence of the positions 3,021 to 3,203, the complementarysequence of the sequence of the positions 3,470 to 4,051, the sequenceof the positions 4,280 to 4,480, and the sequence of the positions 4,520to 4,717, respectively. The amino acid sequences of the G3798, G3797,G3796, G3795, G3794, G3793, and G3792 proteins of the Escherichia coliK5 strain are shown as SEQ ID NOS: 150 to 156, respectively.

The ryjA gene encodes a low molecular weight RNA. The ryjA gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 4,275,950 to 4,276,089 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990).

The soxR and soxS genes are genes encoding a transcriptional controlfactor. The soxR and soxS genes of the Escherichia coli K-12 MG1655strain correspond to the sequence of the positions 4,275,492 to4,275,956, and the complementary sequence of the sequence of thepositions 4,275,083 to 4,275,406 in the genome sequence registered atthe NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2GI: 49175990), respectively. The SoxR and SoxS proteins of the MG1655strain are registered as GenBank accession NP_418487 (versionNP_418487.1 GI:16131889) and GenBank accession NP_418486 (versionNP_418486.1 GI: 16131888), respectively.

The yjcC gene encodes a c-di-GMP-specific phosphodiesterase. The yjcCgene of the Escherichia coli K-12 MG1655 strain corresponds to thesequence of the positions 4,273,494 to 4,275,080 in the genome sequenceregistered at the NCBI database as GenBank accession NC_000913 (VERSIONNC_000913.2 GI: 49175990). The YjcC protein of the MG1655 strain isregistered as GenBank accession NP_418485 (version NP_418485.1 GI:16131887).

The yjcB gene is a gene of unknown function. The yjcB gene of theEscherichia coli K-12 MG1655 strain corresponds to the complementarysequence of the sequence of the positions 4,272,783 to 4,273,064 in thegenome sequence registered at the NCBI database as GenBank accessionNC_000913 (VERSION NC_000913.2 GI: 49175990). The YjcB protein of theMG1655 strain is registered as GenBank accession NP_418484 (versionNP_418484.4 GI: 90111681).

The nucleotide sequence of a region containing the ryjA, soxR, soxS,yjcC, and yjcB genes of the Escherichia coli K5 strain is shown as SEQID NO: 157. In the nucleotide sequence shown as SEQ ID NO: 157, theryjA, soxR, soxS, yjcC, and yjcB genes correspond to the sequence of thepositions 657 to 796, the complementary sequence of the sequence of thepositions 790 to 1,254, the sequence of the positions 1,340 to 1,663,the complementary sequence of the sequence of the positions 1,666 to3,252, and the sequence of the positions 3,682 to 3,963, respectively.The amino acid sequences of the SoxR, SoxS, YjcC, and YjcB proteins ofthe Escherichia coli K5 strain are shown as SEQ ID NOS: 158 to 161,respectively.

The efeU and efeO genes are genes encoding a component of a divalentiron ion transport carrier. The efeU and efeO genes of the Escherichiacoli K-12 MG1655 strain correspond to the sequence of the positions1,080,579 to 1,081,408, and the sequence of the positions 1,081,466 to1,082,593 in the genome sequence registered at the NCBI database asGenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990),respectively. The efeU gene of the MG1655 strain is considered to be apseudogene. The EfeO protein of the MG1655 strain is registered asGenBank accession NP_415537 (version NP_415537.1 GI: 16128982).

The nucleotide sequence of a region containing the efeU and efeO genesof the Escherichia coli K5 strain is shown as SEQ ID NO: 162. In thenucleotide sequence shown as SEQ ID NO: 162, the efeU and efeO genescorrespond to the sequence of the positions 753 to 1,583, and thesequence of the positions 1,641 to 2,768, respectively. The amino acidsequences of EfeU and EfeO proteins of the Escherichia coli K5 strainare shown as SEQ ID NOS: 163 and 164, respectively.

The bacterium of the present invention may have been modified so that,for example, expression of at least the rfaH gene among the genes ofTables 1 to 3 is increased, or expression of at least one or more genesamong the genes of Tables 1 to 3 other than the rfaH gene is increased.The bacterium of the present invention may have been also modified sothat expression of the rfaH gene and expression of one or more kinds ofthe genes of Tables 1 to 3 other than the rfaH gene are increased.Specifically, the bacterium of the present invention may have beenmodified so that, for example, expression of the rfaH gene andexpression is increased of one or more genes such as rbsR, rbsK, rbsB,hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiJ ybiC, ybiB, nusG, pcoR,pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA,mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY,yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB,gsiC, gsiD, yliE, irp2, irp1, bhsA, and ycfS. The bacterium of thepresent invention may have been also modified so that, for example,expression of at least the rpoE gene among the genes of Tables 1 to 3 isincreased. The combination of the genes of Tables 1 to 3 of whichexpression is to be increased is not particularly limited. Examples ofthe combination include, for example, the combinations described inExamples depicted herein.

The methods for increasing gene expression will be described later.Expression of the gene(s) of Tables 1 to 3 may be increased by, forexample, increasing the copy number of a DNA containing the gene(s) ofTables 1 to 3, such as a DNA having the nucleotide sequence shown as SEQID NO: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95, 99,104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or 162.As for the irp gene, the copy number of a DNA containing a part of theirp gene, such as a DNA having the nucleotide sequence shown as SEQ IDNO: 99, may also be increased. Such DNA as mentioned above of which thecopy number is to be increased may be a variant of a DNA having thenucleotide sequence shown as SEQ ID NO: 29, 34, 37, 43, 50, 54, 60, 64,72, 74, 78, 84, 87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132,134, 140, 144, 149, 157, or 162. For variants of DNA, the descriptionsabout conservative variants of the genes mentioned in Tables 1 to 3 canbe similarly applied. Namely, for example, the copy number of a DNAshowing a homology of 90% or more to the nucleotide sequence shown asSEQ ID NOS: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84, 87, 91, 95,99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144, 149, 157, or162 may be increased.

These genes can be obtained by PCR using a chromosome of a strain havingany of these genes as the template, and oligonucleotides produced on thebasis of any of these known gene sequences as the primers.

The genes of Tables 1 to 3 each may be a variant of the genesexemplified above, so long as the variant maintains the originalfunction. Similarly, the proteins encoded by the genes of Tables 1 to 3each may be a variant of the proteins exemplified above, so long as thevariant maintains the original function. Such a variant that maintainsthe original function may be referred to as a “conservative variant”. Inthe present invention, the genes specified with the aforementioned genenames and the proteins specified with names corresponding to the genenames include the genes and proteins exemplified above, respectively,and in addition, conservative variants thereof. Namely, for example, theterm “rpoE gene” includes the rpoE genes exemplified above (i.e. rpoEgenes of the Escherichia coli K-12 MG1655 strain and the Escherichiacoli K5 strain), and in addition, conservative variants thereof.Similarly, for example, the term “RpoE protein” includes the RpoEproteins exemplified above (i.e. RpoE proteins of the Escherichia coliK-12 MG1655 strain and the Escherichia coli K5 strain), and in addition,conservative variants thereof. Examples of the conservative variantsinclude, for example, homologues and artificially modified variants ofthe genes and proteins exemplified above.

The expression “variant maintains the original function” means that thevariant of a gene or protein has a function (such as activity orproperty) corresponding to the function (such as activity or property)of the original gene or protein.

That is, the expression “variant maintains the original function” meansthat, in the case of the genes of Tables 1 to 3, a variant of any of thegenes has a property of increasing heparosan-producing ability of anEscherichia bacterium having heparosan-producing ability when expressionamount of the variant is increased in the bacterium. Furthermore, theexpression “variant maintains the original function” may also mean that,in the case of the genes of Tables 1 to 3, a variant of any of the genesencodes a protein that maintains the original function. That is, thegenes of Tables 1 to 3 may encode a conservative variant of the proteinsexemplified above.

Similarly, the expression “variant maintains the original function”means that, in the case of the proteins encoded by the genes of Tables 1to 3, a variant of any of the proteins has a property of increasingheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount of the variant isincreased in the bacterium. Further, the expression “variant maintainsthe original function” may also mean that, in the case of the proteinsencoded by the genes of Tables 1 to 3, a variant of any of the proteinshas the above-mentioned function of the corresponding protein, forexample, the function of the sigmaE (σ^(E)) in the case of the RpoEprotein.

Whether a variant of a gene or protein has the property of increasingheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium can be confirmed by introducing the gene or a geneencoding the protein into the Escherichia bacterium havingheparosan-producing ability, and confirming whether theheparosan-producing ability is improved or not.

Homologues of the genes of Tables 1 to 3 can be easily obtained frompublic databases by, for example, BLAST search or FASTA search using anyof the nucleotide sequences of the genes exemplified above as a querysequence. Further, homologues of the genes of Tables 1 to 3 can also beobtained by, for example, PCR using a chromosome of a microorganism suchas bacterium as the template, and oligonucleotides prepared on the basisof any of these known gene sequences as the primers.

The genes of Tables 1 to 3 each may encode a protein having any of theaforementioned amino acid sequences including substitution, deletion,insertion, or addition of one or several amino acid residues at one orseveral positions, so long as the protein maintains the originalfunction. For example, the N-terminus and/or C-terminus of the encodedprotein may be extended or shortened. Although the number of “one orseveral” may differ depending on the positions in the three-dimensionalstructure of the protein or the types of amino acid residues,specifically, it is, for example, 1 to 50, 1 to 40, or 1 to 30, 1 to 20,1 to 10, 1 to 5, or 1 to 3.

The aforementioned substitution, deletion, insertion, or addition of oneor several amino acid residues is a conservative mutation that maintainsnormal function of the protein. Typical examples of the conservativemutation are conservative substitutions. The conservative substitutionis a mutation wherein substitution takes place mutually among Phe, Trp,and Tyr, if the substitution site is an aromatic amino acid; among Leu,Ile, and Val, if it is a hydrophobic amino acid; between Gln and Asn, ifit is a polar amino acid; among Lys, Arg, and His, if it is a basicamino acid; between Asp and Glu, if it is an acidic amino acid; andbetween Ser and Thr, if it is an amino acid having a hydroxyl group.Examples of substitutions considered as conservative substitutionsinclude, specifically, substitution of Ser or Thr for Ala, substitutionof Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or Aspfor Asn, substitution of Asn, Glu, or Gln for Asp, substitution of Seror Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln,substitution of Gly, Asn, Gln, Lys, or Asp for Glu, substitution of Profor Gly, substitution of Asn, Lys, Gln, Arg, or Tyr for His,substitution of Leu, Met, Val, or Phe for Ile, substitution of Ile, Met,Val, or Phe for Leu, substitution of Asn, Glu, Gln, His, or Arg for Lys,substitution of Ile, Leu, Val, or Phe for Met, substitution of Trp, Tyr,Met, Ile, or Leu for Phe, substitution of Thr or Ala for Ser,substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp,substitution of His, Phe, or Trp for Tyr, and substitution of Met, Ile,or Leu for Val. Further, such substitution, deletion, insertion,addition, inversion, or the like of amino acid residues as mentionedabove includes a naturally occurring mutation due to an individualdifference, or a difference of species of the organism from which thegene is derived (mutant or variant).

The genes of Tables 1 to 3 each may be a gene encoding a protein showinga homology of, for example, 80% or more, preferably 90% or more, morepreferably 95% or more, still more preferably 97% or more, particularlypreferably 99% or more, to the total amino acid sequence of any of theamino acid sequences mentioned above, so long as the protein maintainsthe original function. In this description, “homology” may mean“identity”.

The genes of Tables 1 to 3 each may also be a DNA that is able tohybridize under stringent conditions with a probe that can be preparedfrom a known gene sequence, for example, a sequence complementary to apartial or entire sequence of any of the aforementioned nucleotidesequences. The “stringent conditions” refer to conditions under which aso-called specific hybrid is formed, and a non-specific hybrid is notformed. Examples of the stringent conditions include those under whichhighly homologous DNAs hybridize to each other, for example, DNAs notless than 80% homologous, not less than 90% homologous, not less than95% homologous, not less than 97% homologous, not less than 99%homologous, hybridize to each other, and DNAs less homologous than theabove do not hybridize to each other, or conditions of washing oftypical Southern hybridization, i.e., conditions of washing once,preferably 2 or 3 times, at a salt concentration and temperaturecorresponding to 1×SSC, 0.1% SDS at 60° C., or 0.1×SSC, 0.1% SDS at 60°C., or 0.1×SSC, 0.1% SDS at 68° C.

As described above, the probe used for the aforementioned hybridizationmay be a part of a sequence that is complementary to a gene. Such aprobe can be prepared by PCR using oligonucleotides prepared on thebasis of a known gene sequence as the primers and a DNA fragmentcontaining any of the genes of Tables 1 to 3 as the template. As theprobe, for example, a DNA fragment having a length of about 300 bp canbe used. When a DNA fragment having a length of about 300 bp is used asthe probe, the washing conditions of the hybridization may be, forexample, 50° C., 2×SSC and 0.1% SDS.

Furthermore, since the degeneracy of codons differs depending on thehost, the genes of Tables 1 to 3 each may be a gene in which anarbitrary codon is replaced with an equivalent codon, so long as theoriginal function is maintained. For example, the genes of Tables 1 to 3each may be modified so that they have optimal codons according to codonusage of the host.

A variant of the genes of Tables 1 to 3 can be obtained by, for example,modifying a coding region of the genes by site-specific mutagenesis sothat a specific site of the encoded protein include substitution,deletion, insertion, or addition of amino acid residues. Further, avariant of the genes of Tables 1 to 3 can also be obtained by theconventionally known mutagenesis. Examples of the mutagenesis includesuch methods as treating a DNA molecule having a nucleotide sequence ofany of the genes of Tables 1 to 3 in vitro with hydroxylamine or thelike, irradiating X-ray or ultraviolet ray on a microorganism such as amicroorganism belonging to Enterobacteriaceae containing any of thegenes of Tables 1 to 3, treating such a microorganism with a mutagensuch as N-methyl-N′-nitro-N-nitrosoguanidine (NTG), ethylmethanesulfonate (EMS), and methyl methanesulfonate (MMS), performingerror prone PCR (Cadwell, R. C., PCR Meth. Appl., 2, 28 (1992)), DNAshuffling (Stemmer, W. P., Nature, 370, 389 (1994)), and StEP-PCR (Zhao,H., Nature Biotechnol., 16, 258 (1998)), and so forth.

<1-3> Method for Increasing Expression of Gene

Hereafter, methods for increasing (rising) expression of a gene will beexplained.

The expression of a gene may be increased 1.5 times or more, 2 times ormore, or 3 times or more, as compared with that of a non-modifiedstrain. Further, the state that “the expression of a gene is increased”includes not only a state that the expression amount of an objectivegene is increased in a strain that inherently expresses the objectivegene, but also a state that the gene is introduced into a strain thatdoes not inherently express the objective gene, and expressed therein.That is, the phrase “the expression of a gene is increased” may alsomean, for example, that an objective gene is introduced into a strainthat does not possess the gene, and is expressed therein. The state that“the expression of a gene is increased” may also be referred to as “theexpression of a gene is enhanced”.

The expression of a gene can be increased by, for example, increasingthe copy number of the gene.

The copy number of a gene can be increased by introducing the gene intothe chromosome of a host. A gene can be introduced into a chromosome by,for example, using homologous recombination (Miller, J. H., Experimentsin Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only onecopy, or two or more copies of a gene may be introduced. For example, byperforming homologous recombination using a sequence which is present inmultiple copies on a chromosome as a target, multiple copies of a genecan be introduced into the chromosome. Examples of such a sequence whichis present in multiple copies on a chromosome include repetitive DNAs,and inverted repeats located at the both ends of a transposon.Alternatively, homologous recombination may be performed by using anappropriate sequence on a chromosome such as a gene unnecessary for theproduction of an objective substance as a target. Homologousrecombination can be performed by, for example, a method using a linearDNA such as Red-driven integration (Datsenko, K. A., and Wanner, B. L.,Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), a method of using aplasmid containing a temperature sensitive replication origin, a methodof using a plasmid capable of conjugative transfer, a method of using asuicide vector not having a replication origin that functions in a host,or a transduction method using a phage. Furthermore, a gene can also berandomly introduced into a chromosome by using a transposon or Mini-Mu(Japanese Patent Laid-open (Kokai) No. 2-109985, U.S. Pat. No.5,882,888, EP 805867 B1).

Introduction of a target gene into a chromosome can be confirmed bySouthern hybridization using a probe having a sequence complementary tothe whole gene or a part thereof, PCR using primers prepared on thebasis of the sequence of the gene, or the like.

Furthermore, the copy number of a gene can also be increased byintroducing a vector containing the gene into a host. For example, thecopy number of a target gene can be increased by ligating a DNA fragmentcontaining the target gene with a vector that functions in a host toconstruct an expression vector of the gene, and transforming the hostwith the expression vector. The DNA fragment containing the target genecan be obtained by, for example, PCR using the genomic DNA of amicroorganism having the target gene as the template. As the vector, avector autonomously replicable in the cell of the host can be used. Thevector is preferably a multi-copy vector. Furthermore, the vectorpreferably has a marker such as an antibiotic resistance gene forselection of transformant. Furthermore, the vector may have a promoterand/or terminator for expressing the introduced gene. The vector may be,for example, a vector derived from a bacterial plasmid, a vector derivedfrom a yeast plasmid, a vector derived from a bacteriophage, cosmid,phagemid, or the like. Specific examples of vector autonomouslyreplicable in Enterobacteriaceae bacteria such as Escherichia coliinclude, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322,pSTV29 (all of these are available from Takara Bio), pACYC184, pMW219(NIPPON GENE), pTrc99A (Pharmacia), pPROK series vectors (Clontech),pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors(QIAGEN), and the broad host spectrum vector RSF1010.

When a gene is introduced, it is sufficient that the gene is expressiblyharbored by the bacterium of the present invention. Specifically, it issufficient that the gene is introduced so that it is expressed undercontrol by a promoter sequence that functions in the bacterium of thepresent invention. The promoter may be a promoter derived from the host,or a heterogenous promoter. The promoter may be the native promoter ofthe gene to be introduced, or a promoter of another gene. As thepromoter, for example, such a stronger promoter as mentioned later mayalso be used.

A terminator for termination of gene transcription may be locateddownstream of the gene. The terminator is not particularly limited solong as it functions in the bacterium of the present invention. Theterminator may be a terminator derived from the host, or a heterogenousterminator. The terminator may be the native terminator of the gene tobe introduced, or a terminator of another gene. Specific examples of theterminator include, for example, T7 terminator, T4 terminator, fd phageterminator, tet terminator, and trpA terminator.

Vectors, promoters, and terminators available in various microorganismsare disclosed in detail in “Fundamental Microbiology Vol. 8, GeneticEngineering, KYORITSU SHUPPAN CO., LTD, 1987”, and those can be used.

Furthermore, when two or more of genes are introduced, it is sufficientthat the genes each are expressibly harbored by the bacterium of thepresent invention. For example, all the genes may be carried by a singleexpression vector or a chromosome. Furthermore, the genes may beseparately carried by two or more expression vectors, or separatelycarried by a single or two or more expression vectors and a chromosome.An operon constituted by two or more genes may also be introduced. Thecase of “introducing two or more genes” include, for example,introducing respective genes coding for two or more kinds of enzymes,introducing respective genes coding for two or more subunitsconstituting a single enzyme, and a combination of the foregoing cases.

The gene to be introduced is not particularly limited so long as itcodes for a protein that functions in the host. The gene to beintroduced may be a gene derived from the host, or may be a heterogenousgene. The gene to be introduced can be obtained by, for example, PCRusing primers designed on the basis of the nucleotide sequence of thegene, and using the genomic DNA of an organism having the gene, aplasmid carrying the gene, or the like as a template. The gene to beintroduced may also be totally synthesized, for example, on the basis ofthe nucleotide sequence of the gene (Gene, 60(1), 115-127 (1987)).

In addition, when a protein functions as a complex consisting of aplurality of subunits, a part or all of the plurality of subunits may bemodified, so long as the activity of the protein is eventuallyincreased. That is, for example, when the activity of a protein isincreased by increasing the expression of a gene, the expression of apart or all of the plurality of genes that code for the subunits may beenhanced. It is usually preferable to enhance the expression of all ofthe plurality of genes coding for the subunits. Furthermore, thesubunits constituting the complex may be derived from a single kind oforganism or two or more kinds of organisms, so long as the complex has afunction of the objective protein. That is, for example, genes of thesame organism coding for a plurality of subunits may be introduced intoa host, or genes of different organisms coding for a plurality ofsubunits may be introduced into a host.

Further, the expression of a gene can be increased by improving thetranscription efficiency of the gene. The transcription efficiency of agene can be improved by, for example, replacing the promoter of the geneon a chromosome with a stronger promoter. The “stronger promoter” meansa promoter providing an improved transcription of a gene compared withan inherently existing wild-type promoter of the gene. Examples ofstronger promoters include, for example, the known high expressionpromoters such as T7 promoter, trp promoter, lac promoter, thr promoter,tac promoter, trc promoter, tet promoter, araBAD promoter, rpoHpromoter, PR promoter, and PL promoter. Furthermore, as the strongerpromoter, a highly-active type of an existing promoter may also beobtained by using various reporter genes. For example, by making the −35and −10 regions in a promoter region closer to the consensus sequence,the activity of the promoter can be enhanced (WO00/18935). Examples ofhighly active-type promoter include various tac-like promoters(Katashkina J I et al., Russian Federation Patent Application No.2006134574) and pnlp8 promoter (WO2010/027045). Methods for evaluatingthe strength of promoters and examples of strong promoters are describedin the paper of Goldstein et al. (Prokaryotic Promoters inBiotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)), and so forth.

Furthermore, the expression of a gene can also be increased by improvingthe translation efficiency of the gene. The translation efficiency of agene can be improved by, for example, replacing the Shine-Dalgarno (SD)sequence (also referred to as ribosome binding site (RBS)) for the geneon a chromosome with a stronger SD sequence. The “stronger SD sequence”means a SD sequence that provides an improved translation of mRNAcompared with the inherently existing wild-type SD sequence of the gene.Examples of stronger SD sequences include, for example, RBS of the gene10 derived from phage T7 (Olins P. O. et al, Gene, 1988, 73, 227-235).Furthermore, it is known that substitution, insertion, or deletion ofseveral nucleotides in a spacer region between RBS and the start codon,especially in a sequence immediately upstream of the start codon(5′-UTR), significantly affects the stability and translation efficiencyof mRNA, and hence, the translation efficiency of a gene can also beimproved by modifying them.

In the present invention, sites that affect the expression of a gene,such as promoter, SD sequence, and spacer region between RBS and thestart codon, may also be collectively referred to as “expression controlregion”. Expression control regions can be identified by using apromoter search vector or gene analysis software such as GENETYX. Theseexpression control regions can be modified by, for example, a method ofusing a temperature sensitive vector, or the Red driven integrationmethod (WO2005/010175).

The translation efficiency of a gene can also be improved by, forexample, modifying codons. In Escherichia coli etc., a clear codon biasexists among the 61 amino acid codons found within the population ofmRNA molecules, and the level of cognate tRNA appears directlyproportional to the frequency of codon usage (Kane, J. F., Curr. Opin.Biotechnol., 6 (5), 494-500 (1995)). That is, if there is a large amountof mRNA containing an excess amount of rare codons, a translationalproblem may arise. According to recent research, it is suggested thatclusters of AGG/AGA, CUA, AUA, CGA, or CCC codons may especially reduceboth the quantity and quality of a synthesized protein. Such a problemoccurs especially at the time of expression of a heterologous gene.Therefore, in the case of heterogenous expression of a gene or the like,the translation efficiency of the gene can be improved by replacing arare codon present in the gene with a synonymous codon more frequentlyused. Codons can be replaced by, for example, the site-specific mutationmethod for introducing an objective mutation into an objective site ofDNA. Examples of the site-specific mutation method include the methodutilizing PCR (Higuchi, R., 61, in PCR Technology, Erlich, H. A. Eds.,Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)),and the method utilizing phage (Kramer, W. and Frits, H. J., Meth. inEnzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154,367 (1987)). Alternatively, a gene fragment in which objective codonsare replaced may be totally synthesized. Frequencies of codons invarious organisms are disclosed in the “Codon Usage Database”(www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292(2000)).

Furthermore, the expression of a gene can also be increased byamplifying a regulator that increases the expression of the gene, ordeleting or attenuating a regulator that reduces the expression of thegene.

Such methods for increasing the gene expression as mentioned above maybe used independently or in any arbitrary combination.

The method for the transformation is not particularly limited, andconventionally known methods can be used. There can be used, forexample, a method of treating recipient cells with calcium chloride soas to increase the permeability thereof for DNA, which has been reportedfor the Escherichia coli K-12 strain (Mandel, M. and Higa, A., J. Mol.Biol., 1970, 53, 159-162), and a method of preparing competent cellsfrom cells which are in the growth phase, followed by transformationwith DNA, which has been reported for Bacillus subtilis (Duncan, C. H.,Wilson, G A. and Young, F. E., Gene, 1977, 1:153-167). Alternatively,there can also be used a method of making DNA-recipient cells intoprotoplasts or spheroplasts, which can easily take up recombinant DNA,followed by introducing a recombinant DNA into the DNA-recipient cells,which is known to be applicable to Bacillus subtilis, actinomycetes, andyeasts (Chang, S. and Choen, S. N., 1979, Mol. Gen. Genet., 168:111-115;Bibb, M. J., Ward, J. M. and Hopwood, O. A., 1978, Nature, 274:398-400;Hinnen, A., Hicks, J. B. and Fink, G R., 1978, Proc. Natl. Acad. Sci.USA, 75:1929-1933). Further, the electric pulse method reported forcoryneform bacteria (Japanese Patent Laid-open (Kokai) No. 2-207791) canalso be used.

An increase in the expression of a gene can be confirmed by confirmingan increase in the transcription amount of the gene, or by confirming anincrease in the amount of a protein expressed from the gene. An increasein the expression of a gene can also be confirmed by confirming anincrease in the activity of a protein expressed from the gene.

An increase of the transcription amount of a gene can be confirmed bycomparing the amount of mRNA transcribed from the gene with that of anon-modified strain such as a wild-type strain or parent strain.Examples of the method for evaluating the amount of mRNA includeNorthern hybridization, RT-PCR, and so forth (Sambrook, J., et al.,Molecular Cloning A Laboratory Manual/Third Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNAmay increase, for example, 1.5 times or more, 2 times or more, or 3times or more, as compared with that of a non-modified strain.

An increase in the amount of a protein can be confirmed by Westernblotting using antibodies (Molecular Cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor (USA), 2001). The amount of theprotein may increase, for example, 1.5 times or more, 2 times or more,or 3 times or more, as compared with that of a non-modified strain.

An increase in the activity of a protein can be confirmed by measuringthe activity of the protein. The activity of the protein may increase,for example, 1.5 times or more, 2 times or more, or 3 times or more, ascompared with that of a non-modified strain.

The aforementioned methods for increasing the expression of a gene canbe used for enhancement of the expression of arbitrary genes such as thegenes of Tables 1 to 3 and genes encoding a protein that participates inheparosan production.

<2> Method for Producing Heparosan

The method for producing heparosan of the present invention includessteps, for example, of culturing the bacterium of the present inventionin a medium to produce and accumulate heparosan in the medium, andcollecting heparosan from the medium.

The medium to be used is not particularly limited, so long as thebacterium of the present invention can proliferate in the medium, andheparosan is produced and accumulated. As the medium, for example, ausual medium used for culture of bacteria can be used. Specific examplesof the medium include, for example, the LB medium (Luria-Bertani medium,containing 10.0 g of Bacto tryptone, 5.0 g of Bacto yeast extract, and5.0 g of NaCl in 1 litter), but are not limited thereto. As the medium,for example, a medium containing carbon source, nitrogen source,phosphorus source, and sulfur source, as well as components selectedfrom other various organic components and inorganic components asrequired can be used. Types and concentrations of the medium componentsmay be arbitrarily determined by those skilled in the art.

The carbon source is not particularly limited, so long as the bacteriumof the present invention can utilize it to generate heparosan. Specificexamples of the carbon source include, for example, saccharides such asglucose, fructose, sucrose, lactose, galactose, xylose, arabinose,blackstrap molasses, starch hydrolysates, and hydrolysates of biomass,organic acids such as acetic acid, fumaric acid, citric acid, succinicacid, and malic acid, alcohols such as glycerol, crude glycerol, andethanol, and aliphatic acids. As the carbon source, a single kind ofcarbon source may be used, or two or more kinds of carbon sources may beused in combination.

Specific examples of the nitrogen source include, for example, ammoniumsalts such as ammonium sulfate, ammonium chloride, and ammoniumphosphate, organic nitrogen sources such as peptone, yeast extract, meatextract, and soybean protein decomposition products, ammonia, and urea.As the nitrogen source, a single kind of nitrogen source may be used, ortwo or more kinds of nitrogen sources may be used in combination.

Specific examples of the phosphate source include, for example,phosphoric acid salts such as potassium dihydrogenphosphate anddipotassium hydrogenphosphate, and phosphoric acid polymers such aspyrophosphoric acid. As the phosphate source, a single kind of phosphatesource may be used, or two or more kinds of phosphate sources may beused in combination.

Specific examples of the sulfur source include, for example, inorganicsulfur compounds such as sulfates, thiosulfates, and sulfites, andsulfur-containing amino acids such as cysteine, cystine, andglutathione. As the sulfur source, a single kind of sulfur source may beused, or two or more kinds of sulfur sources may be used in combination.

Specific examples of other various organic components and inorganiccomponents include, for example, inorganic salts such as sodium chlorideand potassium chloride; trace metals such as iron, manganese, magnesium,and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6,nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleicacids; and organic components containing those such as peptone, casaminoacid, yeast extract, and soybean protein decomposition product. As othervarious organic components and inorganic components, a single kind ofcomponent may be used, or two or more kinds of components may be used incombination.

Further, when an auxotrophic mutant that requires an amino acid or thelike for growth thereof is used, it is preferable to supplement arequired nutrient to the medium. Furthermore, when a gene is introducedby using a vector carrying an antibiotic resistance gene, it ispreferable to add the corresponding antibiotic to the medium.

Culture conditions are not particularly limited, so long as thebacterium of the present invention can proliferate, and heparosan isproduced and accumulated. The culture can be performed with, forexample, usual conditions used for culture of bacteria. The cultureconditions may be appropriately chosen by those skilled in the art.

The culture can be performed, for example, aerobically as aerationculture or shaking culture by using a liquid medium. The culturetemperature may be, for example, 30 to 37° C. The culture period may be,for example, 16 to 72 hours. The culture can be performed as batchculture, fed-batch culture, continuous culture, or a combination ofthese. The culture may be performed as preculture and main culture. Thepreculture may be performed by using, for example, a plate medium orliquid medium.

As a result of culture of the bacterium of the present invention asdescribed above, heparosan is accumulated in the medium.

The method for collecting heparosan from the culture broth is notparticularly limited, so long as heparosan can be collected. Examples ofthe method for collecting heparosan from the culture broth include, forexample, the method described in Examples. Specifically, for example,culture supernatant can be separated from the culture broth, and thenheparosan contained in the supernatant can be precipitated by ethanolprecipitation. The volume of ethanol to be added may be, for example,2.5 to 3.5 times the volume of the supernatant. The solvent used forprecipitating heparosan is not limited to ethanol, and organic solventsmiscible with water in an arbitrary ratio can be used. Examples of suchorganic solvents include methanol, n-propanol, isopropanol, n-butanol,t-butanol, sec-butanol, propylene glycol, acetonitrile, acetone, DMF,DMSO, N-methylpyrrolidone, pyridine, 1,2-dimethoxyethane, 1,4-dioxane,and THF, as well as ethanol. Precipitated heparosan can be dissolvedwith, for example, water in a volume of 2 times the volume of theoriginal supernatant. The collected heparosan may contain suchcomponents as bacterial cells, medium components, moisture, andby-product metabolites of the bacterium, in addition to heparosan.Heparosan may be purified in a desired degree. Purity of heparosan maybe, for example, 30% (w/w) or higher, 50% (w/w) or higher, 70% (w/w) orhigher, 80% (w/w) or higher, 90% (w/w) or higher, or 95% (w/w) orhigher.

Detection and quantification of heparosan can be performed by a knownmethod. Specifically, for example, heparosan can be detected andquantified by the carbazole method. The carbazole method is a techniquewidely used as a quantification method for uronic acid, in which athermal reaction of heparosan and carbazole can be carried out in thepresence of sulfuric acid, and absorption at 530 nm of the reactionmixture provided by the generated color substance can be measured todetect and quantify heparosan (Bitter T. and Muir H. M. (1962) “Amodified uronic acid carbazole reaction”, Analytical Biochemistry,4(4):330-334). Heparosan can also be detected and quantified by, forexample, treating heparosan with heparinase III, which is a heparosandecomposition enzyme, and performing disaccharide composition analysis.

<3> Method for Producing Heparin

Heparin can be produced by using heparosan produced by the bacterium ofthe present invention. That is, the method for producing heparin of thepresent invention is a method for producing heparin comprising culturingthe bacterium of the present invention in a medium to produce andaccumulate heparosan in the medium, chemically and/or enzymaticallytreating the heparosan to produce heparin, and collecting the heparin.Heparin has an anticoagulant activity, and can be used as an ingredientin drug formulations.

The method for producing heparin from heparosan has already beenreported. Specifically, for example, by subjecting heparosan as astarting material to the steps of (1) N-deacetylation, (2) N-sulfation,(3) C5-epimerization, (4) 2-O-sulfation, (5) 6-O-sulfation, and (6)3-O-sulfation, heparin having an anticoagulant activity can be produced(Zhang Z. et al. (2008) “Solution Structures of ChemoenzymaticallySynthesized Heparin and Its Precursors”, J. Am. Chem. Soc.,130(39):12998-13007). The method for producing heparin may furtherinclude a depolymerization step. Such steps as mentioned above forproducing heparin from heparosan are also collectively referred to as“heparin production process”. The implementation order of the steps inthe heparin production process is not particularly limited, so long asheparin having desired properties can be obtained.

When heparosan is present in the medium, the medium may be subjected tothe heparin production process, or heparosan collected from the mediummay be subjected to the heparin production process. Furthermore,heparosan may be subjected to an arbitrary pretreatment, and then may besubjected to the heparin production process. Examples of thepretreatment include, for example, purification, dilution,concentration, drying, dissolution, and so forth. These pretreatmentsmay also be performed in an appropriate combination. For example, aculture broth containing heparosan as it is, or heparosan purified fromsuch a culture broth to a desired extent may be subjected to the heparinproduction process.

The N-deacetylation can be chemically performed by using, for example,sodium hydroxide. The reaction conditions can be appropriatelydetermined by those skilled in the art. For example, conditionsmentioned in the published reference (Kuberan B. et al. (2003)“Chemoenzymatic Synthesis of Classical and Non-classical AnticoagulantHeparan Sulfate Polysaccharides”, J. Biol. Chem., 278(52):52613-52621)can be referred to.

The N-sulfation can be chemically performed by using, for example,sulfur trioxide/trimethylamine complex. The reaction conditions can beappropriately determined by those skilled in the art. For example,conditions mentioned in the published reference (Kuberan B. et al.(2003) “Chemoenzymatic Synthesis of Classical and Non-classicalAnticoagulant Heparan Sulfate Polysaccharides”, J. Biol. Chem.,278(52):52613-52621) can be referred to.

The C5-epimerization can be enzymatically performed by using, forexample, C5-epimerase. The C5-epimerase is not particularly limited solong as a C5-epimerase that can catalyze the epimerization of theglucuronic acid (GlcUA) residue into the iduronic acid (IdoA) residue ischosen. Depending on the order of the C5-epimerization, N-deacetylation,and/or 0-sulfation, a C5-epimerase showing suitable substratespecificity can be chosen and used. The C5-epimerase may be derived fromany origin such as animal, plant, and microorganism. As theC5-epimerase, for example, human C5-epimerase can be used. The reactionconditions can be appropriately determined by those skilled in the art.For example, conditions mentioned in the published reference (Chen J.,et al., “Enzymatic redesigning of biologically active heparan sulfate”,J. Biol. Chem., 2005 December, 30; 280(52):42817-25) can be referred to.

The 2-O-sulfation can be enzymatically performed by using, for example,a 2-O-sulfation enzyme (2-OST). 2-OST is not particularly limited, solong as the chosen 2-OST can catalyze sulfation of the 0-2 position ofthe IdoA residue. Depending on the order of the 2-O-sulfation,N-deacetylation, C5-epimerization, 6-O-sulfation, and/or 3-O-sulfation,2-OST showing suitable substrate specificity can be chosen and used.2-OST may be derived from any origin such as animal, plant, andmicroorganism. As 2-OST, for example, hamster 2-OST can be used. Thereaction conditions can be appropriately determined by those skilled inthe art. For example, conditions mentioned in the published reference(Chen J., et al., “Enzymatic redesigning of biologically active heparansulfate”, J. Biol. Chem., 2005 December, 30; 280(52):42817-25) can bereferred to.

The 6-O-sulfation can be enzymatically performed by using, for example,a 6-O-sulfation enzyme (6-OST). 6-OST is not particularly limited solong as the chosen 6-OST can catalyze sulfation of the 0-6 position ofN-sulfated glucosamine (GlcNS) residue. Depending on the order of the6-O-sulfation, N-deacetylation, C5-epimerization, 2-O-sulfation, and/or3-O-sulfation, 6-OST showing suitable substrate specificity can bechosen and used. 6-OST may be derived from any origin such as animal,plant, and microorganism. As 6-OST, for example, hamster 6-OST-1 ormouse 6-OST-3 can be used. The reaction conditions can be appropriatelydetermined by those skilled in the art. For example, conditionsmentioned in the published reference (Chen J., et al., “Enzymaticredesigning of biologically active heparan sulfate”, J. Biol. Chem.,2005 December, 30; 280(52):42817-25) can be referred to.

The 3-O-sulfation can be enzymatically performed by using, for example,a 3-O-sulfation enzyme (3-OST). 3-OST is not particularly limited solong as 3-OST that can catalyze sulfation of the O-3 position ofN-sulfated and 6-O-sulfated glucosamine residue is chosen. Depending onthe order of the 3-O-sulfation, N-deacetylation, C5-epimerization,2-O-sulfation, and/or 6-O-sulfation, 3-OST showing suitable substratespecificity can be chosen and used. 3-OST may be derived from any originsuch as animal, plant, and microorganism. As 3-OST, for example, mouse3-OST-1 can be used. The reaction conditions can be appropriatelydetermined by those skilled in the art. For example, conditionsmentioned in the published reference (Chen J., et al., “Enzymaticredesigning of biologically active heparan sulfate”, J. Biol. Chem.,2005 December, 30; 280(52):42817-25) can be referred to.

The depolymerization can be performed, for example, by using nitrousacid or by the photolysis method. Degree of the depolymerization is notparticularly limited. The depolymerization may be performed so thatheparin having a molecular weight of, for example, 1000 to 35000 Da isproduced.

The produced heparin can be collected by known methods used forseparation and purification of compounds. Examples of such methodsinclude, for example, ion-exchange resin method, membrane treatment,precipitation, and crystallization. These methods can be used in anappropriate combination. The collected heparin may contain componentssuch as those used for the heparin production process, and moisture, inaddition to heparin. Heparin may be purified in a desired degree. Purityof heparin may be, for example, 30% (w/w) or higher, 50% (w/w) orhigher, 70% (w/w) or higher, 80% (w/w) or higher, 90% (w/w) or higher,or 95% (w/w) or higher.

The obtained heparin can be further fractionated to obtain a lowmolecular weight heparin. Low molecular weight heparin means, forexample, a fraction of a molecular weight of 1000 to 10000 Da (averagemolecular weight, 4000 to 6000 Da). Low molecular weight heparin has anadvantage that it shows less adverse reaction of hemorrhage comparedwith a non-fractionated heparin.

EXAMPLES

Hereafter, the present invention will be more specifically explainedwith reference to Examples.

Example 1: Construction of Heparosan-Producing Strain from Escherichiacoli BL21(DE3) Strain

Construction of Expression Plasmid for kfiABCD Genes of Escherichia coliK5 Strain

From the Escherichia coli K5 strain (ATCC 23506), the kfiABCD genes(kfiABCD operon) were cloned into the pVK9 vector (SEQ ID NO: 1, U.S.Published Patent Application No. 20050196846) to construct a kfiABCDgene expression plasmid, pVK9-kfiABCD.

The details of the construction of the expression plasmid are describedbelow. By PCR using the chromosomal DNA of the Escherichia coli K5strain as the template, as well as the primer KfiABCD-kpnF (SEQ ID NO:2) and primer KfiABCD-xbaR (SEQ ID NO: 3), a DNA fragment containing thekfiABCD genes and an upstream sequence thereof of about 450 bp wasobtained. PrimeStar Polymerase (TaKaRa) was used for PCR, and PCR wasperformed in the reaction composition described in the attachedprotocol. The PCR cycles consisted of 94° C. for 5 minutes, following 30cycles of 98° C. for 5 seconds, 55° C. for 10 seconds, and 72° C. for 8minutes, and final maintenance at 4° C. Further, by PCR using pVK9 asthe template DNA and the oligonucleotides of SEQ ID NOS: 4 and 5 as theprimers, a DNA fragment of pVK9 was obtained. PrimeStar Polymerase(TaKaRa) was used for PCR, and PCR was performed in the reactioncomposition described in the attached protocol. The PCR cycles consistedof 94° C. for 5 minutes, following 30 cycles of 98° C. for 5 seconds,55° C. for 10 seconds, and 72° C. for 6 minutes, and final maintenanceat 4° C. Both the obtained DNA fragments were ligated by using In-Fusion(registered trademark) HD Cloning Kit (Clontech) to construct a kfiABCDgene expression plasmid, pVK9-kfiABCD. A nucleotide sequence containingthe cloned kfiABCD genes and the upstream sequence thereof of about 450bp is shown as SEQ ID NO: 24.

Construction of kfiABCD Gene-Expressing Strain of Escherichia coliBL21(DE3)

The kfiABCD gene expression plasmid, pVK9-kfiABCD, was introduced intothe Escherichia coli BL21(DE3) strain (Life Technologies) byelectroporation (cell 80 μL, 200 Ω, 25 μF, 1.8 kV, cuvette 0.1 mL) toobtain Escherichia coli BL21(DE3)/pVK9-kfiABCD strain. This strain wasspread on the LB agar medium containing 25 μg/mL kanamycin, andprecultured overnight at 37° C. Then, the cells on the plate werescraped and inoculated into 2 mL of a production medium contained in atest tube. Shaking culture was performed at 37° C. for 40 hours, and theculture was finished when glycerol contained in the medium wascompletely consumed.

The composition of the production medium is shown below.

Production Medium: (Concentrations of Components are FinalConcentrations)

Component 1:

Glycerol 10 g/L

Component 2:

MOPS (3-N-morpholino-propanesulphonic acid) 41.9 g/L

Components 3:

Tryptone 8.8 g/L Yeast extract 4.4 g/L Sodium chloride 8.8 g/L

Components 1 and 3 were separately sterilized by autoclaving at 120° C.for 20 minutes, and component 2 was sterilized by filter sterilization.After cooling to room temperature, three of the components were mixed.

Quantification of Polysaccharides by Carbazole Method

The produced polysaccharides were quantified by the carbazole method(Bitter, T. and Murir H. M., Anal. Biochem., 1962, 4:330-334). Theprocedures are shown below.

The culture supernatant was collected from the culture broth(fermentation broth) by centrifugation. To 150 μL of the culturesupernatant, 500 μL of 100% ethanol was added, and the polysaccharidecomponents were precipitated by centrifugation. The obtainedprecipitates were air-dried, and dissolved in 300 μL of 0.2 N aqueoussodium hydroxide solution. The obtained sample (solution, 30 μL) wascalmly added to 150 μL of sulfuric acid containing 0.025 M tetraboronicacid, and the resulting mixture was heated at 100° C. for 10 minutes.After the mixture was cooled to room temperature, 30 μL of a 0.025%carbazole solution (obtained by dissolving 0.125 g of carbazole in 100mL of 100% ethanol) was added. The resulting mixture was heated at 100°C. for 15 minutes, and then cooled to room temperature, and absorbancewas measured at 530 nm. As a result of quantification performed by usinga standard curve prepared with D-glucuronic acid, the concentration ofthe polysaccharides contained in the sample (solution) was calculated tobe 140.5 mg/L in terms of glucuronic acid concentration.

Example 2: Structural Analysis of Produced Polysaccharides

(2-1) Nuclear Magnetic Resonance (NMR) Spectrum Analysis

The fermentation broth obtained in Example 1 was subjected tobactofugation, and the supernatant was filtered through a 0.45 μm MFmembrane. The obtained filtrate (31 g) was concentrated to 1.1 g byusing a UF membrane of 100 KDa (Amicon-15K, 5000 rpm). The concentratewas further washed twice with 40 mL of water. The washed concentrate wasfurther concentrated under reduced pressure in an evaporator, 600 μL ofheavy water was added to the residue to prepare a solution, and then41-NMR measurement was performed.

The analysis conditions are shown below.

(A) Apparatus: AVANCE400 produced by Bruker; 1H, 400 MHz

(B) Solvent: Heavy water

(C) Temperature: Room temperature

(D) Number of times of measurement: 16 times

As a result, there was observed a spectrum of ¹H-NMR (D₂O) includingpeaks of a: 1.9 (methyl proton of N-acetyl group), 3.3-4.5 (methyleneand methine protons of C2 to C6), and 5.3 (methine proton of C1). Thisspectrum was the same as the ¹H-NMR spectrum of heparosan produced byIduron (lot number, B.N.4).

(2-2) Disaccharide Composition Analysis by Liquid Chromatography-MassSpectrometry (LC-MS)

The fermentation broth obtained in Example 1 was subjected tobactofugation, and the supernatant was filtered through a 0.45 μm MFmembrane. The obtained filtrate (40 mL) was concentrated to 4 mL byusing a UF membrane of 100 KDa (Amicon-15K, 5000 rpm). The concentratewas further washed twice with 40 mL of water. To 50 μL of the washedconcentrate, 10 μL of Tris-buffer (200 mM Tris-HCl, 1 M NaCl, 15 mMCaCl₂, adjusted to pH 7 (25° C.) with 35% hydrochloric acid), 10 μL ofheparinase III (0.005 unit/mL, produced by Iduron), and 30 μL of waterwere added, and enzyme treatment was performed at 37° C. for 16 hours.To the obtained enzyme-treated mixture, 900 μL of water was added, andused for LC-MS analysis.

The analysis conditions are shown below:

(A) Apparatus: LC-MS 2010 produced by Shimadzu

(B) Column: UG80 (SCX, Shiseido), 2.0 mm×250 mm, particle size 5 μm

(C) Mobile phase: CH₃CN/10 mM formic acid=8/2

(D) Flow rate: 0.2 mL/minute

(E) Column temperature: 40° C.

(F) Injection volume: 10

(G) UV (PDA): 200 to 600 nm

(H) MS (ESI): 100 to 2000 (positive and negative)

As a result, fragment ions of [m/z]=362 (M+H-H₂O), 380 (M+H), and 418(M+K) were detected at a retention time of 6 minutes. The retention timeand fragment pattern of the enzyme-treated mixture agreed with theretention time and fragment pattern of a ΔGlcUA-GlcNAc standard sample(Heparin disaccharide IV-A sodium salt, Sigma-Aldrich), which is aheparinase decomposition product of heparin and heparan sulfate. Thestructural formula of the ΔGlcUA-GlcNAc standard sample is shown belowas the formula (I).

On the basis of the aforementioned results of NMR and LC-MS, it wasidentified that the polymer component obtained from the culture broth ofthe BL21(DE3)/pVK9-kfiABCD strain was objective heparosan. Therefore,the glucuronic acid concentration multiplied by a coefficient 2.067 wasused as heparosan concentration measured by the carbazole method.

(2-3) Gel Filtration Chromatography (GPC) Analysis

The fermentation broth obtained in Example 1 was subjected tobactofugation, and the supernatant was filtered through a 0.45 μm MFmembrane. The obtained filtrate (31 g) was concentrated to 1.1 g byusing a UF membrane of 100 KDa (Amicon-15K, 5000 rpm). The concentratewas further washed twice with 40 mL of water. GPC measurement of thewashed concentrate was performed.

Analysis conditions are shown below.

(A) Apparatus: HPLC produced by Shimadzu

(B) Column: Asahipak GS520HQ, 7.5 mm×300 mm

(C) Mobile phase: 100 mM KH₂PO₄

(D) Flow rate: 0.6 mL/minute

(E) Column temperature: 40° C.

(F) Injection volume: 20

(G) UV: 200 nm

(H) Molecular weight standard sample: Pullulan (P-82, Showa Denko)

As a result, it was confirmed that retention time (peak top) was 8.3minutes, number average molecular weight (Mn) was 240,000, weightaverage molecular weight (Mw) was 320,000, and Mw/Mn was 1.3.

Example 3: Screening for Factors that Improve Heparosan-ProducingAbility

In this example, screening was performed for factors that improveheparosan-producing ability by introducing a genomic library of theEscherichia coli K5 strain into a heparosan-producing strain.

(3-1) Construction of Escherichia coli BL21(DE3)-Ptac-rfaH/pVK9-kfiABCDStrain

As a heparosan-producing strain to be introduced with the genomiclibrary, Escherichia coli BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strainintroduced with the kfiABCD gene and showing enhanced expression of therfaH gene was constructed in accordance with the following procedures.

A rfaH gene expression-enhanced strain was obtained by replacing thenative promoter region of the rfaH gene on the chromosome with a potenttac promoter (Amann E. et al. (1983) “Vectors bearing a hybrid trp-lacpromoter useful for regulated expression of cloned genes in Escherichiacoli”, Gene, 25(2-3):167-78). The rfaH promoter was replaced with thetac promoter by using the method called “Red-driven integration”, whichwas originally developed by Datsenko and Wanner (“One-step inactivationof chromosomal genes in Escherichia coli K-12 using PCR products”, Proc.Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645). According to thistechnique, a strain in which a DNA fragment amplified by PCR is insertedinto the genomic DNA can be obtained.

First, by PCR using the genomic DNA of the Pantoea ananatis NA1Δc1129strain (WO2010/027022A1) as the template, as well as the primerrfaH-attL Fw (SEQ ID NO: 6) and primer rfaH-Ptac Rv (SEQ ID NO: 7), aDNA fragment for promoter substitution was amplified. PrimeStarPolymerase was used for PCR, and the PCR cycles consisted of 94° C. for5 minutes, following 30 cycles of 98° C. for 5 seconds, 55° C. for 10seconds, and 72° C. for 3 minutes, and final maintenance at 4° C. Theprimer rfaH-attL Fw (SEQ ID NO: 6) shows homology to both a regionlocating upstream from the rfaH gene, and a region adjacent to the genethat imparts kanamycin (km) resistance existing in the genomic DNA ofthe NA1Δc1129 strain. The Km resistance gene kan existing in the genomicDNA of the NA1Δc1129 strain is inserted between the attL and attR genes,which are the attachment sites of λ phage, and the tac promoter (Ptac,SEQ ID NO: 8) is inserted further downstream therefrom in the order ofattL-kan-attR-Ptac. The primer rfaH-Ptac Rv (SEQ ID NO: 7) showshomology to both the rfaH region and a region locating downstream fromthe tac promoter in the genomic DNA of the NA1Δc1129 strain.

Then, into Escherichia coli BL21(DE3)/pKD46 strain obtained byintroducing the plasmid pKD46 having a temperature sensitive replicationorigin (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 2000,97:12:6640-45) into Escherichia coli BL21(DE3) strain (C6000-03, LifeTechnologies), the PCR product obtained above was introduced byelectroporation to attain substitution of the promoter region. Theplasmid pKD46 contains the genes of the λ Red homologous recombinationsystem (γ, β, and exo genes), i.e. a 2,154 nucleotide DNA fragment ofphage (GenBank/EMBL Accession No. J02459, nucleotide positions 31088 to33241), under the control of the arabinose-inducible P_(araB) promoter.The plasmid pKD46 is necessary for integration of the PCR product intothe chromosome of the BL21(DE3) strain. The Escherichia coliBL21(DE3)/pKD46 strain was grown overnight at 30° C. in the LB mediumcontaining ampicillin (100 mg/L). This culture was diluted 100 timeswith the LB medium (100 mL) containing ampicillin and L-arabinose (1mM). The cells were grown at 30° C. with aeration until OD600 becameabout 0.3, then concentrated 100 times, washed 3 times with ice-cooledaqueous glycerol solution (10%), and thereby made into electrocompetentcells. Electroporation was performed by using 70 μl of the competentcells and about 100 ng of the PCR product. After the electroporation,the cells were incubated in 1 mL of the SOC medium (Molecular Cloning ALaboratory Manual, 2nd edition, Sambrook, J. et al., Cold Spring HarborLaboratory Press (1989)) at 37° C. for 2.5 hours, applied to the LB agarmedium, and grown at 37° C. to select Km resistant strains.

The substitution of the tac promoter for the rfaH promoter was confirmedby PCR using the primer rfaH CF (SEQ ID NO: 9) and primer rfaH CR (SEQID NO: 10), which are specific to the nucleotide sequence after thepromoter substitution. PrimeStar Polymerase was used for PCR. The PCRcycles consisted of 94° C. for 5 minutes, following 30 cycles of 98° C.for 5 seconds, 55° C. for 10 seconds, and 72° C. for 2 minutes, andfinal maintenance at 4° C. A strain in which amplification of a DNAfragment of 1.6 kbp could be confirmed was designated asBL21(DE3)-Ptac-rfaH(KmR) strain.

In order to remove the Km resistance marker from theBL21(DE3)-Ptac-rfaH(KmR) strain, plasmid pMW118-int-xis (ampicillinresistant (AmpR)) was introduced (WO2005/010175) to the strain. AmpRclones were grown at 30° C. on an LB agar plate containing 150 mg/L ofampicillin. Several tens of AmpR clones were picked up, and aKm-sensitive strain was selected. By incubating the Km sensitive strainat 42° C. on an LB agar plate, the plasmid pMW118-int-xis was removedfrom the Km-sensitive strain. An obtained Amp sensitive strain wasdesignated as BL21(DE3)-Ptac-rfaH strain. The plasmid pVK9-kfiABCDproduced in Example 1 was introduced into the BL21(DE3)-Ptac-rfaH strainby electroporation to obtain BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain.Culture was performed in test tubes by using the same medium and culturemethod as those of Example 1, and heparosan production amount wasdetermined by the carbazole method. The heparosan production amounts ofthe BL21(DE3)/pVK9-kfiABCD strain of which expression of the rfaH genewas not enhanced, and the BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain ofwhich expression of the rfaH gene was enhanced are shown in Table 4.

TABLE 4 Heparosan production amount of BL21(DE3)- Ptac-rfaH/pVK9-kfiABCDstrain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD 290.4 ± 32.7BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD 506.2 ± 69.9

(3-2) Construction of Genomic Library of Escherichia coli K5 Strain

Fragments of the genomic DNA of the Escherichia coli K5 strain werecloned into the pSTV28 vector (SEQ ID NO: 11, TaKaRa) to constructedgenomic library.

The details of the construction of the genomic library are shown below.The genomic DNA of the Escherichia coli K5 strain (3 μg) was randomlyfragmented by using a DNA fragmentation apparatus (Hydro-Shear, GeneMachine), and fractionated by agarose electrophoresis. A portioncontaining DNAs of about 3 to 5 kb was cut out from the agarose gel, andDNAs were extracted, purified, and then blunt-ended. Then, the genomicDNA fragments were ligated with 50 ng of the plasmid vector pSTV28(TaKaRa) digested with HincII and dephosphorylated with AlkalinePhosphatase (E. coli C75) (TaKaRa). The Escherichia coli HST08 strain(TaKaRa) was transformed with the ligation product by electroporation.Seventy percent or more of the obtained transformants contained insertsof about 3 to 5 kb. The transformants were cultured, and the plasmidswere extracted to obtain a genomic library.

(3-3) Selection of Strains Showing Heparosan-Producing Ability Improvedby Introduction of Genomic Library

The genomic library or pSTV28 as a control was introduced into theBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain by electroporation. One clonewas selected from each of the obtained genomic library transformants,and used to perform fermentative production culture. Media of thefollowing compositions were used for the culture.

Seed Medium: (Concentrations of Components are Final Concentrations)

Tryptone 10 g/L Yeast extract  5 g/L Sodium chloride 10 g/L

The seed medium was sterilized by autoclaving at 120° C. for 20 minutes.

Production Medium: (Concentrations of Components are FinalConcentrations)

Component 1:

Glycerol 10 g/L

Component 2:

MOPS (3-N-morpholino-propanesulphonic acid) 41.9 g/L

Components 3:

Tryptone 8.8 g/L Yeast extract 4.4 g/L Sodium chloride 8.8 g/L

The components 1 and 3 were separately sterilized by autoclaving at 120°C. for 20 minutes, and the component 2 was sterilized by filtersterilization. After cooling to room temperature, three of thecomponents were mixed.

The heparosan production culture was performed according to thefollowing procedures. First, one colony of each transformant wasinoculated into each well of a 96-well plate (MEDISCAN), which contained750 μL of the seed medium, and shaking culture was performed overnightat 37° C. on a shaking machine (Tietech). Then, 20 μL of the seedculture broth was inoculated into 2 mL of the production mediumcontained in a test tube, shaking culture was performed at 37° C. for 30hours, and the culture was terminated when the glycerol in the mediumwas completely consumed. In order to make the cells harbor the plasmids,kanamycin (25 mg/L) and chloramphenicol (25 mg/L) were added to themedium over the whole culture period. Heparosan produced in the mediumwas quantified by the carbazole method (Bitter, T. and Murir H. M.,Anal. Biochem., 1962, 4:330-334). There were isolated clones that showedincreased heparosan accumulation amounts as compared with thesimultaneously cultured control vector (pSTV28)-introduced strain. Inorder to identify the genes inserted into the plasmids contained in theisolated clones, the nucleotide sequences of the inserted DNA fragmentswere determined by using the primer pSTV Fw (SEQ ID NO: 12) and primerpSTV Rv (SEQ ID NO: 13). As a result, it was revealed that therespective plasmids contained rbsBKR-hsrA, glgBX, ybiXIJCB, rcsBD-micF,pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW,ybjIJK-rybB, thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB,gsiCD-yliE, irp (part), bhsA-ycfS, lepB-rnc-era, dapA-gcvR-bcp-hyfA,rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, glmZ-hemYXD, rlmL,artQMJ-rlmC-ybjO, yejOML, rpoS-ygbNML,g3798-g3797-g3796-g3795-g3794-g3793-g3792, ryjA-soxRS-yjcCB, and efeUO.The irp (part) means a part of the irp2 gene and a part of the irp1gene. The nucleotide sequences of the inserted fragments containingrbsBKR-hsrA, glgBX, ybiXIJCB, rcsBD-micF, pcoESR, yhcNO-aaeBAX,g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB,thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp(part), bhsA-ycfS, lepB-rnc-era, dapA-gcvR-bcp-hyfA,rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, glmZ-hemYXD, rlmL,artQMJ-rlmC-ybjO, yejOML, rpoS-ygbNML,g3798-g3797-g3796-g3795-g3794-g3793-g3792, ryjA-soxRS-yjcCB, and efeUOare shown as SEQ ID NOS: 29, 34, 37, 43, 50, 54, 60, 64, 72, 74, 78, 84,87, 91, 95, 99, 104, 107, 111, 116, 121, 124, 128, 132, 134, 140, 144,149, 157, and 162, respectively. From the respective isolated clones,plasmids pSTV28-rbsBKR-hsrA, pSTV28-glgBX, pSTV28-ybiXIJCB,pSTV28-rcsBD-micF, pSTV28-pcoESR, pSTV28-yhcNO-aaeBAX,pSTV28-g1455-alpA-g1453, pSTV28-yrbA-mlaBCDEF-yrbG, pSTV28-norW,pSTV28-ybjIJK-rybB, pSTV28-thrBAL-yjtD-yjjY, pSTV28-fruA-psuK,pSTV28-ytfT-yjfF-fbp, pSTV28-yagU-paoAB, pSTV28-gsiCD-yliE, pSTV28-irp,pSTV28-bhsA-ycfS, pSTV28-lepB-rnc-era, pSTV28-dapA-gcvR-bcp-hyfA,pSTV28-rpoE-nadB-yfiC-srmB, pSTV28-g1414-g1413, pSTV28-nuoEFG,pSTV28-glmZ-hemYXD, pSTV28-rlmL, pSTV28-artQMJ-rlmC-ybjO, pSTV28-yejOML,pSTV28-rpoS-ygbNML, pSTV28-g3798-g3797-g3796-g3795-g3794-g3793-g3792,pSTV28-ryjA-soxRS-yjcCB, and pSTV28-efeUO were extracted.

Example 4: Heparosan Production Using rbsBKR-hsrA, glgBX, ybiXIJCB, andrcsBD-micF Gene Expression-Enhanced Strains (1)

There were constructed respective strains from the Escherichia coliBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain constructed in Example 1 byintroducing thereto pSTV28-rbsBKR-hsrA, pSTV28-glgBX, pSTV28-ybiXIJCB,and pSTV28-rcsBD-micF isolated in Example 3, and pSTV28 as a control.Fermentative production culture was performed with these strains, andamounts of the produced heparosan were compared. The strains werecultured in test tubes in quadruplicate by using the same medium andculture method as those shown in Example 1, and heparosan was quantifiedby the carbazole method. Averages and standard deviations of themeasured heparosan concentrations are shown in Table 5.

TABLE 5 Effect of enhancement of rbsBKR-hsrA, glgBX, ybiXIJCB, ormicF-rcsDB gene expression in BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strainStrain Heparosan (mg/L) BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28 529.6 ±46.3 BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 766.4 ± 156.7 rbsBKR-hsrABL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 679.8 ± 9.7 glgBXBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 753.4 ± 129.2 ybiXIJCBBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 656.7 ± 57.7 micF-rcsDB

Example 5: Heparosan Production Using rbsBKR-hsrA, glgBX, ybiXIJCB, andrcsBD-micF Gene Expression-Enhanced Strains (2)

There were constructed respective strains from the Escherichia coliBL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducingthereto pSTV28-rbsBKR-hsrA, pSTV28-glgBX, pSTV28-ybiXIJCB, andpSTV28-rcsBD-micF isolated in Example 3, and pSTV28 as a control.Fermentative production culture was performed with these strains, andamounts of the produced heparosan were compared. The strains werecultured in test tubes in quadruplicate by using the same medium andculture method as those shown in Example 1, and heparosan was quantifiedby the carbazole method. Averages and standard deviations of themeasured heparosan concentrations are shown in Table 6.

TABLE 6 Effect of enhancement of rbsBKR-hsrA, glgBX, ybiXIJCB, ormicF-rcsDB gene expression in BL21(DE3)/pVK9-kfiABCD strain StrainHeparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pSTV28 145.3 ± 14.3BL21(DE3)/pVK9-kfiABCD/pSTV28-rbsBKR-hsrA 286.9 ± 53.3BL21(DE3)/pVK9-kfiABCD/pSTV28-glgBX 238.7 ± 44.2BL21(DE3)/pVK9-kfiABCD/pSTV28-ybiXIJCB 238.1 ± 55.4BL21(DE3)/pVK9-kfiABCD/pSTV28-micF-rcsDB 167.0 ± 7.0

Example 6: Heparosan Production Using rfaH Gene Expression-EnhancedStrain

(6-1) Construction of Expression Plasmid for rfaH Gene of Escherichiacoli B Strain

The rfaH gene was cloned from the Escherichia coli BL21(DE3) strain intopMIV-Pn1p0-ter to construct a rfaH gene expression plasmid,pMIV-Pn1p0-rfaH. pMIV-Pn1p0-ter contains the potent n1p0 promoter(Pn1p0) and the rrnB terminator, and the promoter and the terminator canfunction as an expression unit of a target gene when the target gene isinserted therebetween. “Pn1p0” means the wild-type promoter of the nlpDgene of the Escherichia coli K-12 strain.

The details of the construction of the expression plasmid are shownbelow. By PCR using the chromosomal DNA of Escherichia coli MG1655 asthe template, as well as the primer P1 (SEQ ID NO: 14) and primer P2(SEQ ID NO: 15), there was obtained a DNA fragment containing thepromoter region of the nlpD gene of about 300 bp (wild-type nlpD genepromoter is henceforth referred to as “Pn1p0”). The sites for therestriction enzymes SalI and PaeI were designed in the 5′ end regions ofthe respective primers. The PCR cycles consisted of 95° C. for 3minutes, following 2 cycles of 95° C. for 60 seconds, 50° C. for 30seconds, and 72° C. for 40 seconds, 25 cycles of 94° C. for 20 seconds,55° C. for 20 seconds, and 72° C. for 15 seconds, and 72° C. for 5minutes as the final cycle. The obtained fragment was treated with SalIand PaeI, and inserted into pMIV-5JS (Japanese Patent Laid-open (Kokai)No. 2008-99668) at the SalI-PaeI site to obtain plasmid pMIV-Pn1p0. Thenucleotide sequence of the PaeI-SalI fragment of the Pn1p0 promoterinserted into this pMIV-Pn1p0 plasmid is as shown as SEQ ID NO: 16.

Then, by PCR using the chromosomal DNA of MG1655 as the template, aswell as the primer P3 (SEQ ID NO: 17) and primer P4 (SEQ ID NO: 18), aDNA fragment (SEQ ID NO: 19) containing about 300 bp of the terminatorregion of the rrnB gene was obtained. The sites for the restrictionenzymes XbaI and BamHI were designed in the 5′ end regions of therespective primers. The PCR cycles consisted of 95° C. for 3 minutes,following 2 cycles of 95° C. for 60 seconds, 50° C. for 30 seconds, and72° C. for 40 seconds, 25 cycles of 94° C. for 20 seconds, 59° C. for 20seconds, and 72° C. for 15 seconds, and 72° C. for 5 minutes as thefinal cycle. The obtained fragment was treated with XbaI and BamHI, andinserted into pMIV-Pn1p0 at the XbaI-BamHI site to obtain plasmidpMIV-Pn1p0-ter.

Then, by PCR using the chromosomal DNA of the Escherichia coli BL21(DE3)strain as the template, as well as the primer rfaH Fw (SEQ ID NO: 20)and primer rfaH Rv (SEQ ID NO: 21), a rfaH gene fragment was obtained.The sites for the restriction enzymes SalI and XbaI were designed in the5′ end regions of the respective primers. PrimeStar Polymerase was usedfor PCR, and the PCR cycles consisted of 94° C. for 5 minutes, following30 cycles of 98° C. for 5 seconds, 55° C. for 10 seconds, and 72° C. for4 minutes, and final maintenance at 4° C. The obtained fragment wastreated with SalI and XbaI, and inserted into pMIV-Pn1p0-ter at theSalI-XbaI site to obtain plasmid pMIV-Pn1p0-rfaH. As described above,there was constructed an rfaH expression unit comprising the nlpDpromoter, rfaH gene, and rrnB terminator connected in this order in thepMIV-5JS vector. The nucleotide sequence of the rfaH gene of theEscherichia coli BL21(DE3) strain cloned in this experiment is shown asSEQ ID NO: 46.

(6-2) Heparosan Production Using rfaH Gene Expression-Enhanced Strain

There were constructed respective strains from the Escherichia coliBL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducingthereto pMIV-Pn1p0-rfaH and pMIV-5JS as a control. Fermentativeproduction culture was performed with these strains, and amounts of theproduced heparosan were compared. The medium, culture method, andquantification method for heparosan were the same as those describedabove. Averages and standard deviations of the measured heparosanconcentrations are shown in Table 7.

TABLE 7 Effect of enhancement of rfaH gene expression inBL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L)BL21(DE3)/pVK9-kfiABCD/pMIV-5JS 376.9 ± 48.3BL21(DE3)/pVK9-kfiABCD/pMIV-Pnlp0-rfaH 857.9 ± 219.1

Example 7: Heparosan Production Using nusG Gene Expression-EnhancedStrain

(7-1) Construction of Expression Plasmid for nusG Gene of Escherichiacoli B Strain

By PCR using the chromosomal DNA of the Escherichia coli BL21(DE3)strain as the template, as well as the primer nusG Fw (SEQ ID NO: 22)and primer nusG Rv (SEQ ID NO: 23), a nusG gene fragment was obtained.The sites for the restriction enzymes SalI and XbaI were designed in the5′ end regions of the respective primers. PrimeStar Polymerase was usedfor PCR, and the PCR cycles consisted of 94° C. for 5 minutes, following30 cycles of 98° C. for 5 seconds, 55° C. for 10 seconds, and 72° C. for4 minutes, and final maintenance at 4° C. The obtained fragment wastreated with SalI and XbaI, and inserted into pMIV-Pn1p0-ter treatedwith the same restriction enzymes at the SalI-XbaI site to obtainplasmid pMIV-Pn1p0-nusG in which the nusG gene was cloned. Thenucleotide sequence of the nusG gene of the Escherichia coli BL21(DE3)strain cloned in this experiment is shown as SEQ ID NO: 48.

(7-2) Heparosan Production Using nusG Gene Expression-Enhanced Strain

There were constructed respective strains from the Escherichia coliBL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducingthereto pMIV-Pn1p0-nusG and pMIV-5JS as a control. Fermentativeproduction culture was performed with these strains, and amounts of theproduced heparosan were compared. The medium, culture method, andquantification method for heparosan were the same as those describedabove. Averages and standard deviations of the measured heparosanconcentrations are shown in Table 8.

TABLE 8 Effect of enhancement of nusG gene expression inBL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L)BL21(DE3)/pVK9-kfiABCD/pMTV-5JS 376.9 ± 48.3BL21(DE3)/pVK9-kfiABCD/pMTV-Pnlp0-nusG 618.1 ± 14.6

Example 8: Heparosan Production Using pcoESR, yhcNO-aaeBAX,g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB,thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, Irp(Part), and bhsA-ycfS Gene Expression-Enhanced Strains (1)

There were constructed respective strains from the Escherichia coliBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strain constructed in Example 1 byintroducing thereto pSTV28-pcoESR, pSTV28-yhcNO-aaeBAX,pSTV28-g1455-alpA-g1453, pSTV28-yrbA-mlaBCDEF-yrbG, pSTV28-norW,pSTV28-ybjIJK-rybB, pSTV28-thrBAL-yjtD-yjjY, pSTV28-fruA-psuK,pSTV28-ytfT-yjfF-fbp, pSTV28-yagU-paoAB, pSTV28-gsiCD-yliE, pSTV28-irp,and pSTV28-bhsA-ycfS isolated in Example 3, and pSTV28 as a control.Fermentative production culture was performed with these strains, andamounts of the produced heparosan were compared. The strains werecultured in test tubes in quadruplicate by using the same medium andculture method as those shown in Example 1, and heparosan was quantifiedby the carbazole method. Averages and standard deviations of themeasured heparosan concentrations are shown in Table 9.

TABLE 9 Effect of enhancement of pcoESR, yhcNO-aaeBAX, g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB, thrBAL-yjtD-yjjY,fruA-psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp (part), orbhsA-ycfS gene expression in BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD strainStrain Heparosan (mg/L) BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28 559.8 ±72.6 BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 692.0 ± 95.1 pcoESRBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 663.3 ± 136.1 yhcNO-aaeBAXBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 843.1 ± 81. 5 g1455-alpA-g1453BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 877.8 ± 137.8yrbA-mlaBCDEF-yrbG BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 741.0 ± 21.1norW BL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 796.0 ± 235.8 ybjIJK-rybBBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 635.1 ± 7.9 thrBAL-yjtD-yjjYBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 826.0 ± 125.1 fruA-psuKBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 872.3 ± 20.1 ytfT-yjfF-fbpBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 690.2 ± 63.1 yagU-paoABBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 804.1 ± 92.9 gsiCD-yliEBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 691.5 ± 92.4 irpBL21(DE3)-Ptac-rfaH/pVK9-kfiABCD/pSTV28- 994.3 ± 124.1 bhsA-ycfS

Example 9: Heparosan Production Using pcoESR, yhcNO-aaeBAX,g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB,thrBAL-yjtD-yjjY, fruA-psuK, ytfT-yjfF-Fbp, yagU-paoAB, gsiCD-yliE, Irp(Part), and bhsA-ycfS Gene Expression-Enhanced Strains (2)

There were constructed respective strains from the Escherichia coliBL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducingthereto pSTV28-pcoESR, pSTV28-yhcNO-aaeBAX, pSTV28-g1455-alpA-g1453,pSTV28-yrbA-mlaBCDEF-yrbG, pSTV28-norW, pSTV28-ybjIJK-rybB,pSTV28-thrBAL-yjtD-yjjY, pSTV28-fruA-psuK, pSTV28-ytfT-yjfF-fbp,pSTV28-yagU-paoAB, pSTV28-gsiCD-yliE, pSTV28-irp, and pSTV28-bhsA-ycfSisolated in Example 3, and pSTV28 as a control. Fermentative productionculture was performed with these strains, and amounts of the producedheparosan were compared. The strains were cultured in test tubes inquadruplicate by using the same medium and culture method as those shownin Example 1, and heparosan was quantified by the carbazole method.Averages and standard deviations of the measured heparosanconcentrations are shown in Table 10.

TABLE 10 Effect of enhancement of pcoESR, yhcNO-aaeBAX,g1455-alpA-g1453, yrbA-mlaBCDEF-yrbG, norW, ybjIJK-rybB,thrBAL-yjtD-yjjY, fruA- psuK, ytfT-yjfF-fbp, yagU-paoAB, gsiCD-yliE, irp(part), or bhsA-ycfS gene expression in BL21(DE3)/pVK9-kfiABCD strainStrain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pSTV28 106.4 ± 12.4BL21(DE3)/pVK9-kfiABCD/pSTV28-pcoESR 260.8 ± 47.8BL21(DE3)/pVK9-kfiABCD/pSTV28-yhcNO-aaeBAX 237.2 ± 36.6BL21(DE3)/pVK9-kfiABCD/pSTV28-g1455-alpA- 225.6 ± 8.5 g1453BL21(DE3)/pVK9-kfiABCD/pSTV28-yrbA- 215.2 ± 14.9 mlaBCDEF-yrbGBL21(DE3)/pVK9-kfiABCD/pSTV28-norW 216.4 ± 36.6BL21(DE3)/pVK9-kfiABCD/pSTV28-ybjIJK-rybB 301.9 ± 46.9BL21(DE3)/pVK9-kfiABCD/pSTV28-thrBAL-yjtD- 327.2 ± 39.8 yjjYBL21(DE3)/pVK9-kfiABCD/pSTV28-fruA-psuK 209.3 ± 8.1BL21(DE3)/pVK9-kfiABCD/pSTV28-ytfT-yjfF- 220.1 ± 18.5 fbpBL21(DE3)/pVK9-kfiABCD/pSTV28-yagU-paoAB 258.0 ± 26.4BL21(DE3)/pVK9-kfiABCD/pSTV28-gsiCD-yliE 323.3 ± 15.5BL21(DE3)/pVK9-kfiABCD/pSTV28-irp 202.0 ± 15.0BL21(DE3)/pVK9-kfiABCD/pSTV28-bhsA-ycfS 225.5 ± 37.4

Example 10: Heparosan Production Using lepB-Rnc-Era, dapA-gcvR-Bcp-hyfA,rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, glmZ-hemYXD, rlmL,artQMJ-rlmC-ybjO, yejOML, rpoS-ygbNML,g3798-g3797-g3796-g3795-g3794-g3793-g3792, ryjA-soxRS-yjcCB, and efeUOGene Expression-Enhanced Strains

There were constructed respective strains from the Escherichia coliBL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducingthereto pSTV28-lepB-rnc-era, pSTV28-dapA-gcvR-bcp-hyfA,pSTV28-rpoE-nadB-yfiC-srmB, pSTV28-g1414-g1413, pSTV28-nuoEFG,pSTV28-glmZ-hemYXD, pSTV28-rlmL, pSTV28-artQMJ-rlmC-ybjO, pSTV28-yejOML,pSTV28-rpoS-ygbNML, pSTV28-g3798-g3797-g3796-g3795-g3794-g3793-g3792,pSTV28-ryjA-soxRS-yjcCB, and pSTV28-efeUO isolated in Example 3, andpSTV28 as a control. Fermentative production culture was performed withthese strains, and amounts of the produced heparosan were compared. Thestrains were cultured in test tubes in quadruplicate by using the samemedium and culture method as those shown in Example 1, and heparosan wasquantified by the carbazole method. Averages and standard deviations ofthe measured heparosan concentrations are shown in Tables 11 and 12.

TABLE 11 Effect of enhancement of lepB-rnc-era, dapA-gcvR-bcp-hyfA,rpoE-nadB-yfiC-srmB, g1414-g1413, nuoEFG, or glmZ-hemYXD gene expressionin BL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L)BL21(DE3)/pVK9-kfiABCD/pSTV28 210.4 ± 23.6BL21(DE3)/pVK9-kfiABCD/pSTV28-lepB-rnc-era 488.7 ± 83.5BL21(DE3)/pVK9-kfiABCD/pSTV28-dapA-gcvR- 379.8 ± 49.1 bcp-hyfABL21(DE3)/pVK9-kfiABCD/pSTV28-rpoE-nadB- 282.9 ± 54.1 yfiC-srmBBL21(DE3)/pVK9-kfiABCD/pSTV28-g1414-g1413 423.9 ± 119.5BL21(DE3)/pVK9-kfiABCD/pSTV28-nuoEFG 428.5 ± 64.6BL21(DE3)/pVK9-kfiABCD/pSTV28-glmZ-hemYXD 604.2 ± 177.5

TABLE 12 Effect of enhancement of rlmL, artQMJ-rlmC-ybjO, yejOML,rpoS-ygbNML, g3798-g3797-g3796-g3795-g3794- g3793-g3792,ryjA-soxRS-yjcCB, or efeUO gene expression in BL21(DE3)/pVK9-kfiABCDstrain Strain Heparosan (mg/L) BL21(DE3)/pVK9-kfiABCD/pSTV28 147.4 ±16.3 BL21(DE3)/pVK9-kfiABCD/pSTV28-rlmL 242.7 ± 45.8BL21(DE3)/pVK9-kfiABCD/pSTV28-artQMJ-rlmC- 243.4 ± 97.9 ybjOBL21(DE3)/pVK9-kfiABCD/pSTV28-yejOML 310.7 ± 40.8BL21(DE3)/pVK9-kfiABCD/pSTV28-rpoS-ygbNML 257.1 ± 56.8BL21(DE3)/pVK9-kfiABCD/pSTV28-g3798-g3797- 251.6 ± 68.5g3796-g3795-g3794-g3793-g3792 BL21(DE3)/pVK9-kfiABCD/pSTV28-ryjA-soxRS-287.5 ± 46.0 yjcCB BL21(DE3)/pVK9-kfiABCD/pSTV28-efeUO 385.7 ± 88.5

Example 11: Heparosan Production Using rpoE Gene Expression-EnhancedStrain

(11-1) Construction of Expression Plasmid for rpoE Gene of Escherichiacoli K5 Strain

The rpoE gene was cloned from the Escherichia coli K5 strain intopMIV-Pn1p8-ter to construct a rpoE gene expression plasmid,pMIV-Pn1p0-rpoE. pMIV-Pn1p0-ter contains the potent n1p8 promoter(Pn1p8), and the promoter and a terminator can function as an expressionunit of a target gene when the target gene is inserted therebetween.“Pn1p8” means a variant promoter of the nlpD gene of the Escherichiacoli K-12 strain.

The details of the construction of the expression vector pMIV-Pn1p8-terare shown below. In order to make the wild-type nlpD promoter (Pn1p0) bea stronger promoter by modifying the −10 region thereof, the −10 regionwas randomized according to the following procedures. The wild-type nlpDpromoter region (FIG. 1, SEQ ID NO: 165) contains two regions presumedto function as a promoter, and they are indicated as Pn1p1 and Pn1p2,respectively, in the drawing. By PCR using the plasmid pMIV-Pn1p0-terconstructed in Example 6 as the template, as well as the primer P1 (SEQID NO: 14) and primer P7 (SEQ ID NO: 166), there was obtained a DNAfragment of the wild-type nlpD promoter (Pn1p0) of which −10 region(−10(Pn1p1)) contained on the 3′ end side was randomized. The PCR cycleconsisted of 95° C. for 3 minutes, following 2 cycles of 95° C. for 60seconds, 50° C. for 30 seconds, and 72° C. for 40 seconds, 25 cycles of94° C. for 20 seconds, 60° C. for 20 seconds, and 72° C. for 15 seconds,and 72° C. for 5 minutes as the final cycle.

In the same manner, by PCR using the plasmid pMIV-Pn1p0-ter as thetemplate, as well as the primer P2 (SEQ ID NO: 15) and primer P8 (SEQ IDNO: 167), there was obtained a DNA fragment of the wild-type nlpDpromoter (Pn1p0) of which −10 region (−10(Pn1p2)) contained on the 5′end side was randomized. The PCR cycle consisted of 95° C. for 3minutes, following 2 cycles of 95° C. for 60 seconds, 50° C. for 30seconds, and 72° C. for 40 seconds, 25 cycles of 94° C. for 20 seconds,60° C. for 20 seconds, and 72° C. for 15 seconds, and 72° C. for 5minutes as the final cycle.

The obtained fragments for the 3′ end side and 5′ end side were ligatedby using the BglII sites designed in the primers P7 and P8 to constructa DNA fragment containing a variant nlpD promoter in full length, ofwhich two −10 regions were randomized. By PCR using this DNA fragment asthe template, as well as the primer P1 and primer P2, the DNA fragmentcontaining the full length of the variant nlpD promoter was amplified.The PCR cycles consisted of 95° C. for 3 minutes, following 2 cycles of95° C. for 60 seconds, 50° C. for 30 seconds, and 72° C. for 40 seconds,12 cycles of 94° C. for 20 seconds, 60° C. for 20 seconds, and 72° C.for 15 seconds, and 72° C. for 5 minutes as the final cycle.

The amplified DNA fragment containing the full length of the variantnlpD promoter was treated with the restriction enzymes SalI and PaeIdesigned in the 5′ end regions of the primers, and inserted into theplasmid pMIV-Pn1p0-ter similarly treated with SalI and PaeI to replacethe wild-type nlpD promoter (Pn1p0) on the plasmid with the variant nlpDpromoter. From plasmids obtained as described above, one having thepromoter sequence shown in FIG. 2 (Pn1p8, SEQ ID NO: 168) was chosen,and designated as pMIV-Pn1p8-ter. The nucleotide sequence of thePaeI-SalI fragment of the Pn1p8 promoter inserted into this plasmid wasas shown as SEQ ID NO: 169.

The details of the construction of the rpoE gene expression plasmid,pMIV-Pn1p8-rpoE, are described below. By PCR using the chromosomal DNAof the Escherichia coli K5 strain as the template, as well as the primerrpoE-SalI Fw (SEQ ID NO: 170) and primer rpoE-xba Rv (SEQ ID NO: 171), aDNA fragment of the rpoE gene was obtained. PrimeStar Polymerase(TaKaRa) was used for PCR, and PCR was performed in the reactioncomposition described in the attached protocol. The PCR cycles consistedof 94° C. for 5 minutes, following 30 cycles of 98° C. for 5 seconds,55° C. for 10 seconds, and 72° C. for 2 minutes, and final maintenanceat 4° C. Further, by PCR using pMIV-Pn1p8-ter as the template DNA, aswell as the oligonucleotides of SEQ ID NOS: 172 and 173 as the primers,a DNA fragment of pMIV-Pn1p8-ter was obtained. PrimeStar Polymerase(TaKaRa) was used for PCR, and PCR was performed in the reactioncomposition described in the attached protocol. The PCR cycles consistedof 94° C. for 5 minutes, following 30 cycles of 98° C. for 5 seconds,55° C. for 10 seconds, and 72° C. for 6 minutes, and final maintenanceat 4° C. Both the obtained DNA fragments were ligated by using In-Fusion(registered trademark) HD Cloning Kit (Clontech) to construct an rpoEgene expression plasmid, pMIV-Pn1p8-rpoE. The nucleotide sequence of thecloned rpoE gene is shown as SEQ ID NO: 174.

(11-2) Heparosan Production Using rpoE Gene Expression-Enhanced Strain

There were constructed respective strains from the Escherichia coliBL21(DE3)/pVK9-kfiABCD strain constructed in Example 1 by introducingthereto pMIV-Pn1p8-rpoE and pMIV-5JS as a control. Fermentativeproduction culture was performed with these strains, and amounts of theproduced heparosan were compared. The medium, culture method, andquantification method for heparosan were the same as those describedabove. Averages and standard deviations of the measured heparosanconcentrations are shown in Table 13.

TABLE 13 Effect of enhancement of rpoE gene expression inBL21(DE3)/pVK9-kfiABCD strain Strain Heparosan (mg/L)BL21(DE3)/pVK9-kfiABCD/pMIV-5JS  96.1 ± 5.8BL21(DE3)/pVK9-kfiABCD/pMIV-Pnlp8-rpoE 183.6 ± 7.8

While the invention has been described in detail with reference to thepreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, heparosan-producing ability ofbacteria can be improved, and heparosan can be efficiently produced.

DESCRIPTION OF SEQUENCE LISTING

SEQ ID NO: 1, Nucleotide sequence of pVK9

SEQ ID NOS: 2 to 7, Primers

SEQ ID NO: 8, Nucleotide sequence of tac promoter

SEQ ID NOS: 9 and 10, Primers

SEQ ID NO: 11, Nucleotide sequence of pSTV28

SEQ ID NOS: 12 to 15, Primers

SEQ ID NO: 16, Nucleotide sequence of PaeI-SalI fragment containingwild-type nlpD promoter (Pn1p0)

SEQ ID NOS: 17 and 18, Primers

SEQ ID NO: 19, Nucleotide sequence of rrnB terminator

SEQ ID NOS: 20 to 23, Primers

SEQ ID NO: 24, Nucleotide sequence of kfiABCD operon of Escherichia coliK5 strain

SEQ ID NO: 25, Amino acid sequence of KfiA protein of Escherichia coliK5 strain

SEQ ID NO: 26, Amino acid sequence of KfiB protein of Escherichia coliK5 strain

SEQ ID NO: 27, Amino acid sequence of KfiC protein of Escherichia coliK5 strain

SEQ ID NO: 28, Amino acid sequence of KfiD protein of Escherichia coliK5 strain

SEQ ID NO: 29, Nucleotide sequence of region containing rbsBKR-hsrAgenes of Escherichia coli K5 strain

SEQ ID NO: 30, Amino acid sequence of RbsB protein of Escherichia coliK5 strain

SEQ ID NO: 31, Amino acid sequence of RbsK protein of Escherichia coliK5 strain

SEQ ID NO: 32, Amino acid sequence of RbsR protein of Escherichia coliK5 strain

SEQ ID NO: 33, Amino acid sequence of HsrA protein of Escherichia coliK5 strain

SEQ ID NO: 34, Nucleotide sequence of region containing glgBX genes ofEscherichia coli K5 strain

SEQ ID NO: 35, Amino acid sequence of GlgB protein of Escherichia coliK5 strain

SEQ ID NO: 36, Amino acid sequence of GlgX protein of Escherichia coliK5 strain

SEQ ID NO: 37, Nucleotide sequence of region containing ybiXIJCB genesof Escherichia coli K5 strain

SEQ ID NO: 38, Amino acid sequence of YbiX protein of Escherichia coliK5 strain

SEQ ID NO: 39, Amino acid sequence of Ybil protein of Escherichia coliK5 strain

SEQ ID NO: 40, Amino acid sequence of YbiJ protein of Escherichia coliK5 strain

SEQ ID NO: 41, Amino acid sequence of YbiC protein of Escherichia coliK5 strain

SEQ ID NO: 42, Amino acid sequence of YbiB protein of Escherichia coliK5 strain

SEQ ID NO: 43, Nucleotide sequence of region containing rcsBD-micF genesof Escherichia coli K5 strain

SEQ ID NO: 44, Amino acid sequence of RcsB protein of Escherichia coliK5 strain

SEQ ID NO: 45, Amino acid sequence of RcsD protein of Escherichia coliK5 strain

SEQ ID NO: 46, Nucleotide sequence of rfaH gene of Escherichia coliBL21(DE3) strain

SEQ ID NO: 47, Amino acid sequence of RfaH protein of Escherichia coliBL21(DE3) strain

SEQ ID NO: 48, Nucleotide sequence of nusG gene of Escherichia coliBL21(DE3) strain

SEQ ID NO: 49, Amino acid sequence of NusG protein of Escherichia coliBL21(DE3) strain

SEQ ID NO: 50, Nucleotide sequence of region containing pcoRSE genes ofEscherichia coli K5 strain

SEQ ID NO: 51, Amino acid sequence of PcoR protein of Escherichia coliK5 strain

SEQ ID NO: 52, Amino acid sequence of PcoS protein of Escherichia coliK5 strain

SEQ ID NO: 53, Amino acid sequence of PcoE protein of Escherichia coliK5 strain

SEQ ID NO: 54, Nucleotide sequence of region containing yhcNO-aaeBAXgenes of Escherichia coli K5 strain

SEQ ID NO: 55, Amino acid sequence of YchN protein of Escherichia coliK5 strain

SEQ ID NO: 56, Amino acid sequence of YchO protein of Escherichia coliK5 strain

SEQ ID NO: 57, Amino acid sequence of AaeB protein of Escherichia coliK5 strain

SEQ ID NO: 58, Amino acid sequence of AaeA protein of Escherichia coliK5 strain

SEQ ID NO: 59, Amino acid sequence of AaeX protein of Escherichia coliK5 strain

SEQ ID NO: 60, Nucleotide sequence of region containing g1455-alpA-g1453genes of Escherichia coli K5 strain

SEQ ID NO: 61, Amino acid sequence of G1455 protein of Escherichia coliK5 strain

SEQ ID NO: 62, Amino acid sequence of AlpA protein of Escherichia coliK5 strain

SEQ ID NO: 63, Amino acid sequence of G1453 protein of Escherichia coliK5 strain

SEQ ID NO: 64, Nucleotide sequence of region containingyrbA-mlaBCDEF-yrbG genes of Escherichia coli K5 strain

SEQ ID NO: 65, Amino acid sequence of YrbA protein of Escherichia coliK5 strain

SEQ ID NO: 66, Amino acid sequence of MlaB protein of Escherichia coliK5 strain

SEQ ID NO: 67, Amino acid sequence of MlaC protein of Escherichia coliK5 strain

SEQ ID NO: 68, Amino acid sequence of MlaD protein of Escherichia coliK5 strain

SEQ ID NO: 69, Amino acid sequence of MlaE protein of Escherichia coliK5 strain

SEQ ID NO: 70, Amino acid sequence of MlaF protein of Escherichia coliK5 strain

SEQ ID NO: 71, Amino acid sequence of YrbG protein of Escherichia coliK5 strain

SEQ ID NO: 72, Nucleotide sequence of region containing norW gene ofEscherichia coli K5 strain

SEQ ID NO: 73, Amino acid sequence of NorW protein of Escherichia coliK5 strain

SEQ ID NO: 74, Nucleotide sequence of region containing ybjIJK-rybBgenes of Escherichia coli K5 strain

SEQ ID NO: 75, Amino acid sequence of YbjI protein of Escherichia coliK5 strain

SEQ ID NO: 76, Amino acid sequence of YbjJ protein of Escherichia coliK5 strain

SEQ ID NO: 77, Amino acid sequence of YbjK protein of Escherichia coliK5 strain

SEQ ID NO: 78, Nucleotide sequence of region containing yjjY-yjtD-thrLABgenes of Escherichia coli K5 strain

SEQ ID NO: 79, Amino acid sequence of YjjY protein of Escherichia coliK5 strain

SEQ ID NO: 80, Amino acid sequence of YjtD protein of Escherichia coliK5 strain

SEQ ID NO: 81, Amino acid sequence of ThrL protein of Escherichia coliK5 strain

SEQ ID NO: 82, Amino acid sequence of ThrA protein of Escherichia coliK5 strain

SEQ ID NO: 83, Amino acid sequence of ThrB protein of Escherichia coliK5 strain

SEQ ID NO: 84, Nucleotide sequence of region containing fruA-psuK genesof Escherichia coli K5 strain

SEQ ID NO: 85, Amino acid sequence of FruA protein of Escherichia coliK5 strain

SEQ ID NO: 86, Amino acid sequence of PsuK protein of Escherichia coliK5 strain

SEQ ID NO: 87, Nucleotide sequence of region containing ytfT-yjfF-fbpgenes of Escherichia coli K5 strain

SEQ ID NO: 88, Amino acid sequence of YtfT protein of Escherichia coliK5 strain

SEQ ID NO: 89, Amino acid sequence of YjfF protein of Escherichia coliK5 strain

SEQ ID NO: 90, Amino acid sequence of Fbp protein of Escherichia coli K5strain

SEQ ID NO: 91, Nucleotide sequence of region containing yagU-paoAB genesof Escherichia coli K5 strain

SEQ ID NO: 92, Amino acid sequence of YagU protein of Escherichia coliK5 strain

SEQ ID NO: 93, Amino acid sequence of PaoA protein of Escherichia coliK5 strain

SEQ ID NO: 94, Amino acid sequence of PaoB protein of Escherichia coliK5 strain

SEQ ID NO: 95, Nucleotide sequence of region containing gsiCD-yliE genesof Escherichia coli K5 strain

SEQ ID NO: 96, Amino acid sequence of GsiC protein of Escherichia coliK5 strain

SEQ ID NO: 97, Amino acid sequence of GsiD protein of Escherichia coliK5 strain

SEQ ID NO: 98, Amino acid sequence of YliE protein of Escherichia coliK5 strain

SEQ ID NO: 99, Nucleotide sequence of region containing a part of irpgene of Escherichia coli K5 strain

SEQ ID NO: 100, Nucleotide sequence of irp2 gene of Escherichia coli K5strain

SEQ ID NO: 101, Amino acid sequence of Irp2 protein of Escherichia coliK5 strain

SEQ ID NO: 102, Nucleotide sequence of irp1 gene of Escherichia coli K5strain

SEQ ID NO: 103, Amino acid sequence of Irp1 protein of Escherichia coliK5 strain

SEQ ID NO: 104, Nucleotide sequence of region containing bhsA-ycfS genesof Escherichia coli K5 strain

SEQ ID NO: 105, Amino acid sequence of BhsA protein of Escherichia coliK5 strain

SEQ ID NO: 106, Amino acid sequence of YcfS protein of Escherichia coliK5 strain

SEQ ID NO: 107, Nucleotide sequence of region containing lepB-rnc-eragenes of Escherichia coli K5 strain

SEQ ID NO: 108, Amino acid sequence of LepB protein of Escherichia coliK5 strain

SEQ ID NO: 109, Amino acid sequence of Rnc protein of Escherichia coliK5 strain

SEQ ID NO: 110, Amino acid sequence of Era protein of Escherichia coliK5 strain

SEQ ID NO: 111, Nucleotide sequence of region containingdapA-gcvR-bcp-hyfA genes of Escherichia coli K5 strain

SEQ ID NO: 112, Amino acid sequence of DapA protein of Escherichia coliK5 strain

SEQ ID NO: 113, Amino acid sequence of GcvR protein of Escherichia coli

K5 strain

SEQ ID NO: 114, Amino acid sequence of Bcp protein of Escherichia coliK5 strain

SEQ ID NO: 115, Amino acid sequence of HyfA protein of Escherichia coliK5 strain

SEQ ID NO: 116, Nucleotide sequence of region containingrpoE-nadB-yfiC-srmB genes of Escherichia coli K5 strain

SEQ ID NO: 117, Amino acid sequence of RpoE protein of Escherichia coliK5 strain

SEQ ID NO: 118, Amino acid sequence of NadB protein of Escherichia coliK5 strain

SEQ ID NO: 119, Amino acid sequence of YfiC protein of Escherichia coliK5 strain

SEQ ID NO: 120, Amino acid sequence of SrmB protein of Escherichia coliK5 strain

SEQ ID NO: 121, Nucleotide sequence of region containing g1414-g1413genes of Escherichia coli K5 strain

SEQ ID NO: 122, Amino acid sequence of G1414 protein of Escherichia coliK5 strain

SEQ ID NO: 123, Amino acid sequence of G1413 protein of Escherichia coliK5 strain

SEQ ID NO: 124, Nucleotide sequence of region containing nuoEFG genes ofEscherichia coli K5 strain

SEQ ID NO: 125, Amino acid sequence of NuoE protein of Escherichia coliK5 strain

SEQ ID NO: 126, Amino acid sequence of NuoF protein of Escherichia coliK5 strain

SEQ ID NO: 127, Amino acid sequence of NuoG protein of Escherichia coliK5 strain

SEQ ID NO: 128, Nucleotide sequence of region containing glmZ-hemYXDgenes of Escherichia coli K5 strain

SEQ ID NO: 129, Amino acid sequence of HemY protein of Escherichia coli

K5 strain

SEQ ID NO: 130, Amino acid sequence of HemX protein of Escherichia coliK5 strain

SEQ ID NO: 131, Amino acid sequence of HemD protein of Escherichia coliK5 strain

SEQ ID NO: 132, Nucleotide sequence of region containing rlmL gene ofEscherichia coli K5 strain

SEQ ID NO: 133, Amino acid sequence of RlmL protein of Escherichia coliK5 strain

SEQ ID NO: 134, Nucleotide sequence of region containingartQMJ-rlmC-ybjO genes of Escherichia coli K5 strain

SEQ ID NO: 135, Amino acid sequence of ArtQ protein of Escherichia coliK5 strain

SEQ ID NO: 136, Amino acid sequence of ArtM protein of Escherichia coliK5 strain

SEQ ID NO: 137, Amino acid sequence of ArtJ protein of Escherichia coliK5 strain

SEQ ID NO: 138, Amino acid sequence of RlmC protein of Escherichia coliK5 strain

SEQ ID NO: 139, Amino acid sequence of YbjO protein of Escherichia coliK5 strain

SEQ ID NO: 140, Nucleotide sequence of region containing yejOML genes ofEscherichia coli K5 strain

SEQ ID NO: 141, Amino acid sequence of YejO protein of Escherichia coliK5 strain

SEQ ID NO: 142, Amino acid sequence of YejM protein of Escherichia coliK5 strain

SEQ ID NO: 143, Amino acid sequence of YejL protein of Escherichia coliK5 strain

SEQ ID NO: 144, Nucleotide sequence of region containing rpoS-ygbNMLgenes of Escherichia coli K5 strain

SEQ ID NO: 145, Amino acid sequence of RpoS protein of Escherichia coliK5 strain

SEQ ID NO: 146, Amino acid sequence of YgbN protein of Escherichia coliK5 strain

SEQ ID NO: 147, Amino acid sequence of YgbM protein of Escherichia coliK5 strain

SEQ ID NO: 148, Amino acid sequence of YgbL protein of Escherichia coliK5 strain

SEQ ID NO: 149, Nucleotide sequence of region containingg3798-g3797-g3796-g3795-g3794-g3793-g3792 genes of Escherichia coli K5strain

SEQ ID NO: 150, Amino acid sequence of G3798 protein of Escherichia coliK5 strain

SEQ ID NO: 151, Amino acid sequence of G3797 protein of Escherichia coliK5 strain

SEQ ID NO: 152, Amino acid sequence of G3796 protein of Escherichia coliK5 strain

SEQ ID NO: 153, Amino acid sequence of G3795 protein of Escherichia coliK5 strain

SEQ ID NO: 154, Amino acid sequence of G3794 protein of Escherichia coliK5 strain

SEQ ID NO: 155, Amino acid sequence of G3793 protein of Escherichia coliK5 strain

SEQ ID NO: 156, Amino acid sequence of G3792 protein of Escherichia coliK5 strain

SEQ ID NO: 157, Nucleotide sequence of region containingryjA-soxRS-yjcCB genes of Escherichia coli K5 strain

SEQ ID NO: 158, Amino acid sequence of SoxR protein of Escherichia coliK5 strain

SEQ ID NO: 159, Amino acid sequence of SoxS protein of Escherichia coliK5 strain

SEQ ID NO: 160, Amino acid sequence of YjcC protein of Escherichia coliK5 strain

SEQ ID NO: 161, Amino acid sequence of YjcB protein of Escherichia coliK5 strain

SEQ ID NO: 162, Nucleotide sequence of region containing efeUO genes ofEscherichia coli K5 strain

SEQ ID NO: 163, Amino acid sequence of EfeU protein of Escherichia coliK5 strain

SEQ ID NO: 164, Amino acid sequence of EfeO protein of Escherichia coliK5 strain

SEQ ID NO: 165, Nucleotide sequence of wild-type nlpD promoter (Pn1p0)

SEQ ID NOS: 166 and 167, Primers

SEQ ID NO: 168, Nucleotide sequence of variant nlpD promoter (Pn1p8)

SEQ ID NO: 169, Nucleotide sequence of PaeI-SalI fragment containingvariant nlpD promoter (Pn1p8)

SEQ ID NOS: 170 to 173, Primers

SEQ ID NO: 174, Nucleotide sequence of rpoE gene of Escherichia coli K5strain

The invention claimed is:
 1. A method for producing heparin, the methodcomprising: A) culturing an Escherichia coli bacterium having aheparosan-producing ability in a medium to produce and accumulateheparosan in the medium; B) treating the heparosan using chemicalmethods, enzymatic methods, or both chemical and enzymatic methods toproduce heparin; and C) collecting the heparin; wherein the bacteriumhas been modified so that expression is increased of an Escherichia colirpoE gene; wherein the bacterium has been further modified so thatexpression of an Escherchia coli heparosan biosynthesis enzyme gene isincreased.
 2. The method according to claim 1, wherein the bacterium hasbeen further modified so that expression of an Escherichia coli rfaHgene is increased.
 3. The method according to claim 2, wherein thebacterium has been further modified so that expression is increased ofan Escherichia coli nusG gene.
 4. The method according to claim 1,wherein said expression is increased by increasing the copy number ofthe gene(s), modifying a gene expression control sequence of thegene(s), or both.
 5. The method according to claim 3, wherein: the rfaHgene is a DNA comprising the nucleotide sequence of SEQ ID NO: 46, or aDNA comprising a nucleotide sequence having an identity of 90% or higherto the nucleotide sequence of SEQ ID NO: 46 and is able to increaseheparosan-producing ability of an Escherichia bacterium havingheparosan-producing ability when expression amount thereof is increasedin the bacterium; the nusG gene is a DNA comprising the nucleotidesequence of SEQ ID NO: 48, or a DNA comprising a nucleotide sequencehaving an identity of 90% or higher to the nucleotide sequence of SEQ IDNO: 48 and is able to increase heparosan-producing ability of anEscherichia bacterium having heparosan-producing ability when expressionamount thereof is increased in the bacterium; the rpoE gene is a DNAcomprising the complementary sequence of the nucleotide sequence ofpositions 355 to 930 of SEQ ID NO: 116, or a DNA comprising a nucleotidesequence having an identity of 90% or higher to the complementarysequence of the nucleotide sequence of positions 355 to 930 of SEQ IDNO: 116 and is able to increase heparosan-producing ability of anEscherichia bacterium having heparosan-producing ability when expressionamount thereof is increased in the bacterium.